Compositions and Methods for Enhancing Immune Responses to Vaccines

ABSTRACT

The disclosure provides adjuvants, immunogenic compositions, and methods useful for vaccination and immune response. In particular, the disclosure provides a class of adjuvants comprising cationic lipid:co-lipid mixtures and methods for delivering formulated compositions.

CROSS REFERENCE TO OTHER APPLICATIONS

This application claims priority benefit under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 60/939,702, titled: “ADJUVANTCOMPOSITIONS AND METHODS FOR ENHANCING IMMUNE RESPONSES TOPOLYNUCLEOTIDE-BASED VACCINES”, filed May 23, 2007; and U.S. ProvisionalPatent Application No. 61/044,338, titled: COMPOSITIONS AND METHODS FORENHANCING IMMUNE RESPONSES TO VACCINES”, filed Apr. 11, 2008, thedisclosure of each are hereby incorporated by reference in theirentirety for all purposes. This application also incorporates byreference the following documents: U.S. patent application Ser. No.08/097,266, filed on Jul. 23, 1993, now U.S. Pat. No. 5,399,163; U.S.patent application Ser. No. 07/920,106, filed Jul. 24, 1992, now U.S.Pat. No. 5,383,851; and U.S. Pat. No. 7,105,574.

FIELD OF THE DISCLOSURE

The disclosure provides cationic-lipids and their use as adjuvants,immunogenic compositions, and methods useful for vaccination. Thedisclosure also provides compounds, compositions and methods useful forenhancing immune responses, especially the humoral immune response ofvertebrates to vaccines. In particular, the disclosure provides adjuvantcompositions of cytofectin:co-lipid mixtures, wherein the cytofectin isa compound of formula I or II, including(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminiumbromide (GAP-DMORIE). Also disclosed are adjuvant compositions whereinthe adjuvant is GAP-DMORIE, and the co-lipid is1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE), i.e., theVaxfectin® adjuvant.

BACKGROUND OF THE DISCLOSURE

In the late 1980s, it was discovered that direct intramuscular (i.m.)injection of lipid-DNA complexes results in measurable proteinexpression, and that “naked” plasmid DNA (pDNA) is taken up andexpressed in muscle to a greater extent than lipid-DNA complexes(Feigner, Scientific American, 276(6), 102-106 (1997)). Later, it wasshown that cationic lipid-protein mixtures afforded greater immuneresponses after i.m. injection than protein alone (Brunel, Vaccine 17,2192-2203 (1999).

One of the first applications of pDNA injection technology was theinduction of an immune response. In 1991, it was first reported thatmice could be immunized against HIV gp120 by i.m. vaccination with gp120plasmid DNA (Felgner et al., Nature, 349, 351-352 (1991)), and that micecould be protected from a lethal challenge of influenza virus after DNAimmunization with influenza nucleoprotein (NP) antigen. Protectionobtained after immunization with the highly conserved NP antigenextended across 2 different viral strains (Ulmer et al., CurrentOpinions In Immunology, 8, 531-536 (1996)). Numerous publications in thefield of polynucleotide-based vaccination followed thereafter (e.g.,Boyer et al., J. Med. Primatology, 25(3), 242-250 (1996); Boyer et al.,Nature Medicine, 3(5), 526-532 (1997); Davis et al., Vaccine, 15(8),849-852 (1997); Wang et al., Vaccine, 15(8), 821-825 (1997); Agadjanyanet al., Current Topics In Microbiology And Immunology, 226, 175-192(1998); Heppell et al., Fish & Shellfish Immunology, 8(4), 271-286(1998); Lodmell et al., Nature Medicine, 4(8), 949-952 (1998);Vanderzanden et al., Virology, 246(1), 134-144 (1998)).

A major problem frequently encountered in the course ofpolynucleotide-based vaccination is insufficient or suboptimal humoralresponse. Often, the antigens or immunogens encoded by thepolynucleotide are expressed in vivo, but they are not sufficientlyimmunogenic to raise the antibody titer in the organism to sufficientlevels to provide protection against subsequent challenge and/or tomaintain the potential for generating therapeutically active antibodylevels over extended time periods. To obtain a stronger humoral and/orcellular response, it is common to administer such vaccines in animmunogenic composition containing an adjuvant, a material whichenhances the immune response of the patient to the vaccine. Adjuvantsare useful generally for improving the immune response of an organism toa particular immunogen and are commonly included in vaccine compositionsto increase the amount of antibodies produced and/or to reduce thequantity of immunogen and the frequency of administration.

A variety of adjuvants have been reported to effect differing levels ofimmune response enhancement to polynucleotide-based vaccination.Examples of such adjuvant materials include semi-synthetic bacterialcell wall-derived mono-phosphoryl lipid A (Sasaki, S., et al., Infectionand Immunity 65(9), 3250-3258 (1997)), small molecule immunostimulators(Sasaki, S., et al., Clin. Exp. Immunol. 111, 30-35 (1998)), andsaponins (Sasaki, S., et al., J. Virology 72(6), 4391-4939 (1998)). Theimmune response from i.m. pDNA vaccination has also been enhancedthrough the use of cationic lipids (Ishii, N., et al., Aids Res. Hum.Retroviruses 13(16), 1421-1428 (1997)), Okada, E., et al., J. Immunology159, 3638-3647 (1997); Yokoyama, M., et al., FEMS Immunol. Med.Microbiol. 14, 221-230 (1996); Gregoriadis, G., et al., FEBS Letters402, 107-110 (1997); Gramzinski, R. A., et al., Molecular Medicine 4,109-118 (1998); Klavinskis, L. S., et al., Vaccine 15(8), 818-820(1997); Klavinskis, L. S., et al., J Immunology 162, 254-262 (1999);Etchart, N., et al., J. Gen. Virology 78, 1577-1580 (1997); Norman, J.,et al., in Methods in Molecular Medicine, Vol. 29; DNA Vaccines: Methodsand Protocols, D. B. Lowrie and R. Whalen, eds., Chapter 17, pp. 185-196(1999)). Cationic lipids were originally studied to enhance delivery ofpDNA into cells in vitro; however, further development has led tosuccessful specific applications of protein delivery in vivo (Wheeler,C. J., et al., Proc. Nail. Acad. Sci. USA 93, 11454-11459 (1996);Stephan, D. J., et al., Human Gene Therapy 7, 1803-1812 (1996);DeBruyne, L. A., et al., Gene Therapy 5, 1079-1087 (1998)). Accordingly,such cationic lipids may be useful for vaccine applications by enhancingdelivery of the pDNA into the cells responsible for giving rise to thehumoral arm of the immune response, thereby increasing antibody titerlevels.

Commonly used adjuvants, such as Alum, show low levels of immuneresponse enhancement for vaccination (typically less than 3-fold) andpossess undesirable toxicological and manufacturing profiles. Inaddition, cationic lipids used previously for vaccination show only lowlevels of humoral enhancement. Thus, there is a need for more adjuvantcompositions useful for enhancing the immune response of vertebrates toimmunization, especially to pDNA vaccination.

BRIEF SUMMARY OF THE DISCLOSURE

The present disclosure provides compounds, adjuvant and immunogeniccompositions, and methods of using these compounds and compositions forthe vaccination of a vertebrate, to help protect the vertebrate from adisease, to treat a diseased vertebrate, or both. More specifically thepresent disclosure provides a class of cationic lipids that are usefulfor enhancing the immune response of vertebrates to immunization,especially in response to pDNA, polypeptide, peptide, nucleic acid,polysaccharide, or inactivated (protein-based) vaccines derived fromwhole virus particle vaccination.

In certain embodiments, the present disclosure provides methods forimmunizing a vertebrate by administering to the vertebrate a compositioncomprising a polynucleotide that encodes for an immunogen, wherein thepolynucleotide is complexed with an adjuvant composition comprising acationic lipid compound of formula I or II:

or an enantiomer, diastereomer, or a pharmaceutically acceptable salt orsolvate thereof, wherein R¹, R², R³, L₁, L₂, Q, Q′ and T are asdescribed herein, one or more counter ions, and one or more co-lipids.

In other embodiments, the composition comprises one or more co-lipids.The immunogen-encoding polynucleotide, upon incorporation into the cellsof the vertebrate, produces an immunologically effective amount of animmunogen (e.g., an immunogenic protein). The adjuvant composition ofthe present disclosure enhances the immune response of the vertebrate tothe immunogen.

In another aspect, the present disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipid compounds and one ormore co-lipids, which adjuvant composition is useful for enhancing thehumoral immune response of a vertebrate to an immunogen. In someembodiments, the adjuvant composition comprises the cationic lipidcompound of formula I or II, and one or more co-lipids. In otherembodiments, the co-lipid is a neutral lipid such as, for example, aphosphatidylethanolamine including but not limited to1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE), and/or1,2-dimyristoyl-glycero-3-phosphoethanolamine (DMPE), or adialkylglycerol such as 1,2-di-O-phytanyl-sn-glycerol (DPYG) or azwitterionic co-lipid such as DPyRIE carboxylate, ordiphytanoylphosphatidylglycerol.

In another aspect, the present disclosure provides immunogeniccompositions comprising one or more immunogens and an adjuvantcomposition comprising the cationic lipid compound of formula I or II,and one or more co-lipids. In certain embodiments, the source of theimmunogen is an immunogen-encoding polynucleotide, such as in the caseof a pDNA vaccine. In some embodiments, the pDNA or polynucleotide maybe complexed with an adjuvant composition comprising the cationic lipidcompound of formula I or II, and one or more co-lipids.

In another aspect, the present disclosure provides methods forimmunizing a vertebrate by administering to the vertebrate animmunogenic composition comprising a complex of one or moreimmunogen-encoding polynucleotides and the cationic lipid compound offormula I or II in an amount sufficient to generate an immune responseto the encoded immunogen. In some embodiments, the immunogeniccomposition further includes one or more co-lipids such as, for example,DOPE and/or DPyPE.

The present disclosure further provides compositions and methods usefulfor enhancing the humoral immune response of a vertebrate to apolynucleotide-based vaccine, through the use of the compounds offormula I or II. Elevation of antibody levels is particularlyadvantageous in applications where antibody levels from theimmunogen-encoding polynucleotide alone are sub-optimal. In a relatedadvantage, if the desired level of antibodies is produced with a givendose of pDNA, the amount of pDNA necessary to reach the predeterminedantibody titer level can be reached using a lower pDNA dose. For pDNAvaccination applications, this advantage is important because acceptablevaccination volumes, coupled with functional limits on the concentrationof pDNA, define an upper limit on a given vaccine dose. This advantageis particularly beneficial for vaccines containing multiple plasmids,each of which must be present in sufficient quantity to elicit an immuneresponse to its particular transgene.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate the preferred embodiments of the presentinvention, and together with the description serve to explain theprinciples of the invention.

In the Drawings:

FIG. 1 is a bar graph illustrating enhanced humoral immune responsesobtained with cytofectin:co-lipid vaccine formulations delivered eitherIM or ID with needle & syringe.

FIG. 2 consists of three graphs (2A, 2B and 2C) comparing humoral immuneresponses obtained in rabbits with unformulated pDNA vaccine injected IMwith needle & syringe, or delivered either IM or ID with a needle-freeinjection device Biojector®2000 (B2000), to Vaxfectin®-formulatedvaccine delivered either IM or ID with Biojector®2000.

FIG. 2A shows anti-gB geometric mean titers (GMT) obtained three weeksafter a single injection, whereas FIG. 2B shows anti-gB titers obtainedthree weeks after boost injections. FIG. 2C shows more detailed timecourses of humoral responses in rabbits immunized with unformulated pDNAinjected IM with needle vs. responses obtained in rabbits immunized withVaxfectin®-formulated pDNA delivered with Biojector®2000 device.

FIG. 3 consists of two bar graphs (3A and 3B) comparing humoral immuneresponses obtained in rabbits with pDNA vaccine formulated withpoloxamer CRL1005+BAK to responses obtained with cytofectin:co-lipidvaccine formulations delivered either IM with needle & syringe, or IDwith Biojector®2000 device.

FIG. 3A shows anti-gB geometric mean titers (GMT) obtained three weeksafter boost injections. FIG. 3B shows more detailed time courses ofhumoral responses in rabbits immunized with poloxamer formulations vs.responses obtained in rabbits immunized with DMRIE:DOPE-formulated pDNAdelivered ID with Biojector®2000 device (B2000).

FIG. 4 consists of three graphs (4A, 4B and 4C) comparing IM vs. IDdelivery routes in rabbits using Vaxfectin®-formulated pDNA andBiojector®2000 device vs. IM injections performed with needle & syringe.

FIGS. 4A & 4B show 6-week anti-gB geometric mean titers (GMT) obtainedin rabbits vaccinated with various doses of VCL6365 pDNA formulated withVaxfectin®, and immunized either via IM or ID route. FIG. 4C shows moredetailed time courses of humoral responses in rabbits immunized withVaxfectin®formulations delivered ID with B2000 device vs. responsesobtained with Vaxfectinφformulation injected IM with needle & syringe.

FIG. 5 consists of four graphs (5A, 5B, 5C and 5D) illustrating humoralimmune responses and survival in influenza challenge model in miceimmunized either with intradermal injections with needle & syringe, orby delivering the vaccine epidermally/intradermally with OMNI device.

FIG. 5A and FIG. 5B show average anti-M2 and anti-NP serum titersobtained in mice on Day 35, respectively. FIG. 5C and FIG. 5D showsurvival and average body mass of vaccinated mice challenged withinfluenza virus on Day 48, respectively.

FIG. 6 consists of five graphs (6A, 6B, 6C, 6D and 6E) illustratingimmune responses in mice vaccinated with the same dose ofVaxfectin®-formulated pDNA injected either IM or ID with needle &syringe, or delivered epidermally/intradermally with OMNI device, anddemonstrates the survival of immunized mice in influenza challengemodel.

FIG. 6A and FIG. 6B show NP-specific CD8+ and CD4+ T-cell responses onDay 21, respectively. FIG. 6C and FIG. 6D show average anti-NP andanti-M2 serum titers on Day 21, respectively. FIG. 6E illustratessurvival of vaccinated mice challenged with influenza virus on Day 14.

DETAILED DESCRIPTION OF THE DISCLOSURE

It will be apparent to one skilled in the art, in view of the followingdetailed description and the claims appended hereto, that varioussubstitutions and modifications may be made to the present disclosurewithout departing from the scope of the disclosure.

DEFINITIONS

Abbreviations used herein have their conventional meaning within thechemical and biological arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e. unbranched) or branched chain,or cyclic hydrocarbon radical, or combination thereof, which may befully saturated, mono- or polyunsaturated and can include di- andmultivalent radicals, having the number of carbon atoms designated (i.e.C₁-C₁₀ means one to ten carbons). Examples of saturated hydrocarbonradicals include, but are not limited to, groups such as methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. Alkyl groups which arelimited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkyl, as exemplified, but not limited,by CH₂CH₂CH₂CH₂—. Typically, an alkyl (or alkylene) group will have from1 to 24 carbon atoms, with those groups having 10 or fewer carbon atomsbeing preferred in the present disclosure. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingeight or fewer carbon atoms.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of atleast one carbon atom and at least one heteroatom selected from thegroup consisting of O, N, P, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N, P and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,&H═CH—N(CH₃)—CH₃, O—CH₃, —O—CH₂—CH₃, and —CN. Up to two heteroatoms maybe consecutive, such as, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.Similarly, the term “heteroalkylene” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— andCH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxo,alkylenedioxo, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)OR′—represents both —C(O)OR′ and —R′OC(O)—. As described above, heteroalkylgroups, as used herein, include those groups that are attached to theremainder of the molecule through a heteroatom, such as —C(O)R′,—C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO₂R′. Where “heteroalkyl” isrecited, followed by recitations of specific heteroalkyl groups, such as—NR′R″ or the like, it will be understood that the terms heteroalkyl and—NR′R″ are not redundant or mutually exclusive. Rather, the specificheteroalkyl groups are recited to add clarity. Thus, the term“heteroalkyl” should not be interpreted herein as excluding specificheteroalkyl groups, such as —NR′R″ or the like.

An “alkylesteryl,” as used herein, refers to a moiety having the formulaR′—C(O)O—R″, wherein R′ is an alkylene moiety and R″ is an alkyl moiety.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. The terms “cycloalkylene”and “heterocycloalkylene” refer to the divalent derivatives ofcycloalkyl and heterocycloalkyl, respectively.

The term “cycloalkyl” or “cycloalkylalkyl” also refers to a 3 to 7membered cycloalkyl group attached to the remainder of the molecule viaan unsubstituted alkylene group. Recitation of a specific number ofcarbon atoms (e.g., C₁-C₁₀ cycloalkylalkyl) refers to the number ofcarbon atoms in the alkylene group.

The term “heterocycloalkyl” or “heterocycloalkylalkyl” also refers to a3 to 7 membered heterocycloalkyl group attached to the remainder of themolecule via an unsubstituted alkylene group. Recitation of a specificnumber of carbon atoms (e.g., C₁-C₁₀ hetero-cycloalkylalkyl) refers tothe number of carbon atoms in the alkylene group.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (preferably from 1 to 3 rings) which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a carbon or heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolinyl, 5-isoquinolinyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolinyl, and 6-quinolinyl. Substituents for each of above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below. The terms “arylene” and“heteroarylene” refer to the divalent derivatives of aryl andheteroaryl, respectively.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxo, arylthioxo, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

The term “oxo” as used herein means an oxygen that is double bonded to acarbon atom.

Each of above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl, and“heterocycloalkyl”, “aryl,” “heteroaryl” as well as their divalentradical derivatives) are meant to include both substituted andunsubstituted forms of the indicated radical. Preferred substituents foreach type of radical are provided below.

Substituents for alkyl, heteroalkyl, cycloalkyl, heterocycloalkylmonovalent and divalent derivative radicals (including those groupsoften referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —C(O)NR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)OR′,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R′″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g.,aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl,alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of thedisclosure includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R′″ groupswhen more than one of these groups is present. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From above discussion of substituents, one of skill inart will understand that the term “alkyl” is meant to include groupsincluding carbon atoms bound to groups other than hydrogen groups, suchas haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g., —C(O)CH₃,—C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for alkyl radicals above,exemplary substituents for aryl and heteroaryl groups (as well as theirdivalent derivatives) are varied and are selected from, for example:halogen, —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO₂R′, —C(O)NR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″,—NR″C(O)OR′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′,—S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂,fluoro(C₁-C₄)alkoxo, and fluoro(C₁-C₄)alkyl, in a number ranging fromzero to the total number of open valences on aromatic ring system; andwhere R′, R″, R′″ and R″″ are preferably independently selected fromhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound of the disclosure includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present.

Two of the substituents on adjacent atoms of aryl or heteroaryl ring mayoptionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, wherein Tand U are independently —NR—, —O—, —CRR′— or a single bond, and q is aninteger of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′—(C″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″ and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl.

As used herein, the term “heteroatom” or “ring heteroatom” is meant toinclude oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

The compounds of the present disclosure may exist as salts. The presentdisclosure includes such salts. Examples of applicable salt formsinclude hydrochlorides, hydrobromides, sulfates, methanesulfonates,nitrates, maleates, acetates, citrates, fumarates, tartrates (e.g.,(+)-tartrates, (−)-tartrates or mixtures thereof including racemicmixtures, succinates, benzoates and salts with amino acids such asglutamic acid. These salts may be prepared by methods known to thoseskilled in art. Also included are base addition salts such as sodium,potassium, calcium, ammonium, organic amino, or magnesium salt, or asimilar salt. When compounds of the present disclosure containrelatively basic functionalities, acid addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired acid, either neat or in a suitable inert solvent.Examples of acceptable acid addition salts include those derived frominorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogen-phosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived organicacids like acetic, propionic, isobutyric, maleic, malonic, benzoic,succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike. Certain specific compounds of the present disclosure contain bothbasic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

Certain compounds of the present disclosure can exist in unsolvatedforms as well as solvated forms, including hydrated forms. In general,the solvated forms are equivalent to unsolvated forms and areencompassed within the scope of the present disclosure. Certaincompounds of the present disclosure may exist in multiple crystalline oramorphous forms. In general, all physical forms are equivalent for theuses contemplated by the present disclosure and are intended to bewithin the scope of the present disclosure.

Certain compounds of the present disclosure possess asymmetric carbonatoms (optical centers) or double bonds; the enantiomers, racemates,diastereomers, tautomers, geometric isomers, stereoisometric forms thatmay be defined, in terms of absolute stereochemistry, as (R)- or (S)-or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the present disclosure. The compounds ofthe present disclosure do not include those which are known in art to betoo unstable to synthesize and/or isolate. The present disclosure ismeant to include compounds in racemic and optically pure forms.Optically active (R)- and (S)-, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques. When the compounds described herein contain olefinic bondsor other centers of geometric asymmetry, and unless specified otherwise,it is intended that the compounds include both E and Z geometricisomers.

Where two groups are “optionally joined together to form a ring,” thetwo groups are covalently bonded together with the atom or atoms towhich the two groups are joined to form a substituted or unsubstitutedaryl, a substituted or unsubstituted heteroaryl, a substituted orunsubstituted cycloalkyl, or a substituted or unsubstitutedheterocycloalkyl ring.

The terms “arylalkyl,” “heteroarylalkyl,” “cycloalkyl-alkyl,” and“heterocycloalkyl-alkyl,” as used herein, refer to an aryl, heteroaryl,cycloalkyl and heterocycloalkyl, respectively, attached to the remainderof the molecule via an alkylene group. Where an “arylalkyl,”“heteroarylalkyl,” “cycloalkyl-alkyl,” or “heterocycloalkyl-alkyl” issubstituted, one or more substituent moieties may be covalently bondedto the alkylene moiety and/or the aryl, heteroaryl, cycloalkyl andheterocycloalkyl moieties, respectively. A “C₁-C₂₀” arylalkyl,heteroarylalkyl, cycloalkyl-alkyl, or heterocycloalkyl-alkyl, aremoieties in which a C₁-C₂₀ alkylene links an aryl, heteroaryl, C₄-C₈cycloalkyl, and 4 to 8 membered heterocycloalkyl, respectively, to theremainder of the molecule. A “C₁-C₈” arylalkyl, heteroarylalkyl,cycloalkyl-alkyl, or heterocycloalkyl-alkyl, are moieties in which aC₁-C₈ alkylene links an aryl, heteroaryl, C₅-C₇ cycloalkyl, and 5 to 7membered heterocycloalkyl, respectively, to the remainder of themolecule.

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

-   -   (A) —OH, —NH₂, —SH, —CN, —CF₃, oxy, halogen, unsubstituted        alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,        unsubstituted heterocycloalkyl, unsubstituted aryl,        unsubstituted heteroaryl, and    -   (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and        heteroaryl, substituted with at least one substituent selected        from:        -   (i) oxy, —OH, —NH₂, —SH, —CN, —CF₃, halogen, unsubstituted            alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,            unsubstituted heterocycloalkyl, unsubstituted aryl,            unsubstituted heteroaryl, and        -   (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,            and heteroaryl, substituted with at least one substituent            selected from:            -   (a) oxy, —OH, —NH₂, —SH, —CN, —CF₃, halogen,                unsubstituted alkyl, unsubstituted heteroalkyl,                unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, unsubstituted                heteroaryl, and            -   (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,                aryl, or heteroaryl, substituted with at least one                substituent selected from oxy, —OH, —NH₂, —SH, —CN,                —CF₃, halogen, unsubstituted alkyl, unsubstituted                heteroalkyl, unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, and unsubstituted                heteroaryl.

A “size-limited substituent” or “size-limited substituent group,” asused herein means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₄-C₈cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 4 to 8 membered heterocycloalkyl.

A “lower substituent” or “lower substituent group,” as used herein meansa group selected from all of the substituents described above for a“substituent group,” wherein each substituted or unsubstituted alkyl isa substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₅-C₇ cycloalkyl, and each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7membered heterocycloalkyl.

The term “higher alkyl” refers to those alkyl groups having at least sixcarbon atoms. The term “lower alkyl” refers to those alkyl groups havingfrom one to five carbon atoms.

The term “tautomer,” as used herein, refers to one of two or morestructural isomers which exist in equilibrium and which are readilyconverted from one isomeric form to another.

It will be apparent to one skilled in the art that certain compounds ofthis disclosure may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the disclosure.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of thedisclosure.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of this disclosure.

The compounds of the present disclosure may also contain unnaturalproportions of atomic isotopes at one or more of atoms that constitutesuch compounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (3H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe present disclosure, whether radioactive or not, are encompassedwithin the scope of the present disclosure.

The term “pharmaceutically acceptable salts” is meant to include saltsof active compounds which are prepared with relatively nontoxic acids orbases, depending on the particular substituent moieties found on thecompounds described herein. When compounds of the present disclosurecontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentdisclosure contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al., “Pharmaceutical Salts”, Journal ofPharmaceutical Science, 1977, 66, 1-19). Certain specific compounds ofthe present disclosure contain both basic and acidic functionalitiesthat allow the compounds to be converted into either base or acidaddition salts.

In addition to salt forms, the present disclosure relates to compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentdisclosure. Additionally, prodrugs can be converted to the compounds ofthe present disclosure by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present disclosure when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

The terms “a,” “an,” or “a(n)”, when used in reference to a group ofsubstituents herein, mean at least one. For example, where a compound issubstituted with “an” alkyl or aryl, the compound is optionallysubstituted with at least one alkyl and/or at least one aryl. Moreover,where a moiety is substituted with an R substituent, the group may bereferred to as “R-substituted.” Where a moiety is R-substituted, themoiety is substituted with at least one R substituent and each Rsubstituent is optionally different.

Description of compounds of the present disclosure are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, physiological conditions.

The term “treating” refers to any indicia of success in the treatment,amelioration or prevention of an injury, pathology or condition,including any objective or subjective parameter such as abatement;remission; diminishing of symptoms or making the injury, pathology orcondition more tolerable to the patient; slowing in the rate ofdegeneration or decline; making the final point of degeneration lessdebilitating; improving a patient's physical or mental well-being. Thetreatment, amelioration or prevention of symptoms can be based onobjective or subjective parameters; including the results of a physicalexamination, neuropsychiatric exams, and/or a psychiatric evaluation.

The symbol

denotes the point of attachment of a moiety to the remainder of themolecule.

Cationic Lipid Compounds of Formula I and Formula II

The present disclosure is directed to the polynucleotide-based andpolypeptide-based immunization of a vertebrate, to protect from or treata vertebrate with a disease condition. The present disclosure includesthe use of cationic lipids, especially the cationic lipid compound offormula I or II in adjuvants, immunogenic compositions, and methods forimmunizing a vertebrate, especially with polynucleotide-based immunogen.

The adjuvant composition of the present disclosure includes one or morecationic lipids and, in some embodiments, one or more co-lipids. In oneembodiment, the cationic lipid is a compound of formula I or II:

wherein R¹, R², R³, L₁, L₂, Q, Q′ and T are as described below.

In one aspect, the disclosure provides compounds having formula I:

or an enantiomer, diastereomer, or a pharmaceutically acceptable salt orsolvate thereof, wherein:

R¹ and R¹ are each independently substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl,substituted or unsubstituted arylalkyl, substituted or unsubstitutedheteroarylalkyl, or substituted or unsubstituted carboxyalkyl, whereinR¹ and R² are each optionally independently substituted with 1 to 5 R¹⁶groups;

L₁ and L₂ are each independently a direct bond, O, NH, N(C₁-C₆ alkyl),or S(O)_(m), wherein m is an integer from 0 to 2;

n is an integer from 0 to 6;

Q is independently -Z₁N⁺Z₂- or -Z₁P⁺Z₂-;

Z₁ and Z₂ are each independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, substituted or unsubstitutedcarboxyalkyl, or substituted or unsubstituted —(CH₂)_(m)—R³ wherein m isan integer from 1 to 6, and wherein Z₁ and Z₂ are each optionallyindependently substituted with 1 to 5 R¹⁶ groups;

T is substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl, substituted or unsubstitutedarylalkyl, substituted or unsubstituted heteroarylalkyl, or substitutedor unsubstituted carboxyalkyl; wherein Q is optionally independentlysubstituted with 1 to 5 R¹⁶ groups; and

R³ is independently —OR⁴, —S(O)_(m)R⁵, —NR⁶R⁷, —N⁺R⁸R⁹R¹⁰, —PR¹¹R¹², or—P⁺R¹³R¹⁴R¹⁵;

R⁴ and R⁵ are each independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, or substituted or unsubstitutedcarboxyalkyl, wherein R⁴ and R⁵ are each optionally independentlysubstituted with 1 to 5 R¹⁶ groups;

R⁶ and R⁷ are each independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, substituted or unsubstitutedcarboxyalkyl, or R⁶ and R⁷, together with the N atom to which they areattached, form substituted or unsubstituted heteroaryl or substituted orunsubstituted heterocycloalkyl, wherein R⁶ and R⁷ are each optionallyindependently substituted with 1 to 5 R¹⁶ groups;

R⁸, R⁹ and R¹⁰ are each independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl,substituted or unsubstituted heteroarylalkyl, or substituted orunsubstituted carboxyalkyl, or R⁸ is as described, and R⁹ and R¹⁰,together with the N atom to which they are attached, form substituted orunsubstituted heteroaryl or substituted or unsubstitutedheterocycloalkyl, wherein R⁸, R⁹ and R¹⁰ are each optionallyindependently substituted with 1 to 5 R¹⁶ groups;

R¹¹, R¹² and R¹³ are each independently substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, substituted or unsubstitutedcarboxyalkyl, or R¹¹ is as described, and R¹² and R¹³, together with theP atom to which they are attached, form substituted or unsubstitutedheterocycloalkyl, wherein R¹¹, R¹² and R¹³ are each optionallyindependently substituted with 1 to 5 R¹⁶ groups;

R¹⁶ is hydrogen, halogen, nitro, cyano, hydroxyl, alkyl, cycloalkyl,perfluoroalkyl, heteroalkyl, heterocycloalkyl, aryl, arylalkyl,heteroaryl, heteroarylalkyl, —(CH₂)_(j)CN, —(CH₂)_(j)OR¹⁷,—(CH₂)_(j)C(O)R¹⁷, —(CH₂)_(j)C(O)OR¹⁷, —(CH₂)_(j)NR¹⁸R¹⁹,—(CH₂)_(j)C(O)NR¹⁸R¹⁹, —(CH₂)_(j)OC(O)NR¹⁸R¹⁹, —(CH₂)_(j)NR²⁰C(O)R⁷,—(CH₂)_(j)NR²⁰C(O)OR¹⁷, —(CH₂)_(j)N²⁰C(O)NR¹⁸R¹⁹, —(CH₂)_(j)S(O)_(m)R²¹,—(CH₂)_(j)S(O)₂NR¹⁸R¹⁹, or —(CH₂)_(j)NR²⁰S(O)₂R²¹, wherein each j isindependently an integer from 0 to 6, and each m is independently aninteger from 0 to 2;

R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹ are each independently hydrogen, substitutedor unsubstituted alkyl, substituted or unsubstituted cycloalkyl,perfluoroalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted —O-aryl, substituted or unsubstitutedarylalkyl, substituted or unsubstituted heteroaryl, substituted orunsubstituted —O-heteroaryl, or substituted or unsubstitutedheteroarylalkyl, or

R¹⁷, R²⁰ and R²¹ are as described above, and R¹⁸ and R¹⁹, together withthe N atom to which they are attached, form substituted or unsubstitutedheteroaryl, or substituted or unsubstituted heterocycloalkyl;

with the proviso that the compound of formula I is not GAP-DMORIE; and

one or more counter ions.

In another aspect, the disclosure provides an adjuvant compositioncomprising a mixture of one or more cationic lipids having formula I:

or an enantiomer, diastereomer, or a pharmaceutically acceptable salt orsolvate thereof, wherein:

R¹ and R² are each independently substituted or unsubstituted alkyl(includes alkenyl and alkynyl), substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl, substituted or unsubstitutedarylalkyl, substituted or unsubstituted heteroarylalkyl, or substitutedor unsubstituted carboxyalkyl, wherein R¹ and R² are each optionallyindependently substituted with 1 to 5 R¹⁶ groups;

L₁ and L₂ are each independently a direct bond, O, NH, N(C₁-C₆ alkyl),Se, Te, or S(O)_(m), wherein m is an integer from 0 to 2;

n is an integer from 0 to 6;

Q is independently -Z₁N⁺Z₂- or -Z₁P⁺Z₂-;

Z₁ and Z₂ are each independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, substituted or unsubstitutedcarboxyalkyl, or substituted or unsubstituted —(CH₂)_(m)—R³ wherein m isan integer from 1 to 6, and wherein Z₁ and Z₂ are each optionallyindependently substituted with 1 to 5 R¹⁶ groups;

T is substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl, substituted or unsubstitutedarylalkyl, substituted or unsubstituted heteroarylalkyl, or substitutedor unsubstituted carboxyalkyl; wherein Q is optionally independentlysubstituted with 1 to 5 R¹⁶ groups; and

R³ is independently —OR⁴, —S(O)_(m)R⁵, —NR⁶R⁷, —N⁺R⁸R⁹R¹⁰, —PR¹¹R¹², or—P⁺R¹³R¹⁴R¹⁵;

R⁴ and R⁵ are each independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, or substituted or unsubstitutedcarboxyalkyl, wherein R⁴ and R⁵ are each optionally independentlysubstituted with 1 to 5 R¹⁶ groups;

R⁶ and R⁷ are each independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, substituted or unsubstitutedcarboxyalkyl, or R⁶ and R⁷, together with the N atom to which they areattached, form substituted or unsubstituted heteroaryl or substituted orunsubstituted heterocycloalkyl, wherein R⁶ and R⁷ are each optionallyindependently substituted with 1 to 5 R¹⁶ groups;

R⁸, R⁹ and R¹⁰ are each independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl,substituted or unsubstituted heteroarylalkyl, or substituted orunsubstituted carboxyalkyl, or R⁸ is as described, and R⁹ and R¹⁰,together with the N atom to which they are attached, form substituted orunsubstituted heteroaryl or substituted or unsubstitutedheterocycloalkyl, wherein R⁸, R⁹ and R¹⁰ are each optionallyindependently substituted with 1 to 5 R¹⁶ groups;

R¹¹, R¹² and R¹³ are each independently substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, substituted or unsubstitutedcarboxyalkyl, or R¹¹ is as described, and R¹² and R¹³, together with theP atom to which they are attached, form substituted or unsubstitutedheterocycloalkyl, wherein R¹¹, R¹² and

R¹³ are each optionally independently substituted with 1 to 5 R¹⁶groups;

R¹⁶ is hydrogen, halogen, nitro, cyano, hydroxyl, alkyl, cycloalkyl,perfluoroalkyl, heteroalkyl, heterocycloalkyl, aryl, arylalkyl,heteroaryl, heteroarylalkyl, —(CH₂)_(j)CN, —(CH₂)_(j)OR¹⁷,—(CH₂)_(j)C(O)R¹⁷, —(CH₂)_(j)C(O)OR¹⁷, —(CH₂)_(j)NR¹⁸R¹⁹,—(CH₂)_(j)C(O)NR¹⁸R¹⁹, —(CH₂)_(j)OC(O)NR¹⁸R¹⁹, —(CH₂)_(j)NR²⁰C(O)R¹⁷,—(CH₂)_(j)NR²⁰C(O)OR¹⁷, —(CH₂)_(j)N²⁰C(O)NR¹⁸R¹⁹, —(CH₂)_(j)S(O)_(m)R²¹,—(CH₂)_(j)S(O)₂NR¹⁸R¹⁹, or —(CH₂)_(j)NR²⁰S(O)₂R²¹, wherein each j isindependently an integer from 0 to 6, and each m is independently aninteger from 0 to 2;

with the proviso that the compound of formula I is not GAP-DMORIE;

one or more counter ions, and

one or more co-lipids.

In another aspect, the disclosure provides an adjuvant composition,wherein:

R¹ and R² are each independently substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, or substituted or unsubstitutedalkynyl;

L₁ and L₂ are each independently O or NH;

n is 1; and

Q is independently -Z₁N⁺Z₂-.

In another aspect, the disclosure provides an adjuvant composition,wherein R¹ and R² are each independently substituted or unsubstituted(C₁-C₂₀)alkyl, substituted or unsubstituted (C₂-C₂₀)alkenyl, orsubstituted or unsubstituted (C₂-C₂₀)alkynyl.

In another aspect, the disclosure provides an adjuvant composition,wherein R¹ and R² are each independently:

In another aspect, the disclosure provides an adjuvant composition,wherein:

Z₁ and Z₂ are each independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, or substituted or unsubstitutedcarboxyalkyl; and

T is substituted or unsubstituted alkyl.

In another aspect, the disclosure provides an adjuvant composition,wherein:

Z₁ and Z₂ are each independently (C₁-C₆)alkyl; and

T is independently (C₁-C₆)alkyl.

In another aspect, the disclosure provides an adjuvant composition,wherein:

Z₁ and Z₂ are each independently substituted or unsubstituted—(CH₂)_(m)—R³;

T is substituted or unsubstituted alkyl; and

R³ is independently —NR⁶R⁷ or —N⁺R⁸R⁹R¹⁰.

In another aspect, the disclosure provides an adjuvant composition,wherein:

T is independently (C₁-C₆)alkyl;

R³ is independently —NR⁵R⁷; and

R⁶ and R⁷ are each independently substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, or R⁶ and R⁷, together withthe N atom to which they are attached, form substituted or unsubstitutedheteroaryl or substituted or unsubstituted heterocycloalkyl.

In another aspect, the disclosure provides an adjuvant composition,wherein R⁶ and R⁷ are each independently substituted or unsubstituted(C₁-C₆)alkyl.

In another aspect, the disclosure provides an adjuvant composition,wherein R⁶ and R⁷, together with the N atom to which they are attached,form substituted or unsubstituted pyrrolyl, substituted or unsubstitutedimidazolyl, substituted or unsubstituted pyrrolidinyl, substituted orunsubstituted pyrazolinyl, substituted or unsubstituted piperidinyl,substituted or unsubstituted piperazinyl, substituted or unsubstitutedmorpholinyl, or substituted or unsubstituted thiomorpholinyl.

In another aspect, the disclosure provides an adjuvant composition,wherein:

T is independently (C₁-C₆)alkyl;

R³ is independently —N⁺R⁸R⁹R¹⁰; and

R⁸, R⁹ and R¹⁰ are each independently substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, or R⁸ is as described,and R⁹ and R¹⁰, together with the N atom to which they are attached,form substituted or unsubstituted heteroaryl or substituted orunsubstituted heterocycloalkyl.

In another aspect, the disclosure provides an adjuvant composition,wherein R⁸, R⁹ and R¹⁰ are each independently substituted orunsubstituted (C₁-C₆)alkyl.

In another aspect, the disclosure provides an adjuvant composition,wherein R⁹ and R¹⁰, together with the N atom to which they are attached,form substituted or unsubstituted pyrrolyl, substituted or unsubstitutedimidazolyl, substituted or unsubstituted pyrrolidinyl, substituted orunsubstituted pyrazolinyl, substituted or unsubstituted piperidinyl,substituted or unsubstituted piperazinyl, substituted or unsubstitutedmorpholinyl, or substituted or unsubstituted thiomorpholinyl.

In another aspect, the disclosure provides an adjuvant composition,wherein the counter ion is negatively charged.

In another aspect, the disclosure provides an adjuvant composition,wherein the negatively charged counter ion is F⁻, Cl⁻, Br⁻, I⁻, CH₃COO⁻,CF₃COO⁻.

In another aspect, the disclosure provides an adjuvant composition,wherein the co-lipid is a neutral lipid.

In another aspect, the disclosure provides an adjuvant composition,wherein the neutral lipid is a phosphatidylethanolamine, and/or aphosphatidylcholine, and/or a mono-, di-, or trialkylglycerol, and/or amono-, di-, or triacylglycerol, and/or a zwitterionic co-lipid such asDPyRIE carboxylate, or phosphatidylinositol, fatty acid, mono-, di-, ortriacylglycerol, lysophosphatidyl-ethanolamine, lysophosphatidylcholine

In another aspect, the disclosure provides an adjuvant composition,wherein the phosphatidylethanolamine is1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and/or1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE), and/or1,2-dimyristoyl-glycero-3-phosphoethanolamine (DMPE).

In another aspect, the disclosure provides an adjuvant composition,wherein the phosphatidylethanolamine is1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE).

In another aspect, the disclosure provides an adjuvant composition,wherein the compound of formula I and the co-lipid ratio is from about9:1 to about 1:9.

In another aspect, the disclosure provides an adjuvant composition,wherein the compound of formula I and the co-lipid are in molar ratio offrom about 4:1 to about 1:4.

In another aspect, the disclosure provides an adjuvant composition,wherein the compound of formula I and the co-lipid are in molar ratio offrom about 2:1 to about 1:2.

In another aspect, the disclosure provides an adjuvant composition,wherein the compound of formula I and the co-lipid are in molar ratio ofabout 1:1.

In another aspect, the disclosure provides an adjuvant composition,wherein the compound of formula I and DPyPE are in molar ratio of fromabout 2:1 to about 1:2.

In another aspect, the disclosure provides an adjuvant composition,wherein the compound of formula I and DPyPE are in molar ratio of about1:1.

In another aspect, the disclosure provides an adjuvant composition,wherein the compound of formula I has formulae:

In another aspect, the disclosure provides methods for immunizing avertebrate comprising administering into a tissue or cavity of thevertebrate an immunogenic composition comprising (a) one or moreantigenic polypeptides, immunogenic polypeptides or polysaccharides inan amount sufficient to generate an immune response to the one or moreantigenic polypeptides, immunogenic polypeptides or polysaccharides, and(b) the adjuvant compositions disclosed herein.

In another aspect, the disclosure provides methods for immunizing avertebrate, wherein the immunogen-encoding polynucleotide, uponincorporation into the cells of the vertebrate, produces animmunologically effective amount of an immunogen (e.g., an immunogenicprotein).

In another aspect, the disclosure provides methods for immunizing avertebrate, wherein the adjuvant composition disclosed herein enhancesthe immune response of the vertebrate to the immunogen.

In another aspect, the disclosure provides immunogenic compositionscomprising one or more immunogens and the adjuvant compositionsdisclosed herein.

In another aspect, the disclosure provides immunogenic compositionscomprising one or more immunogens and the adjuvant compositionsdisclosed herein, wherein the immunogen is one or more antigenicpolypeptides, immunogenic polypeptides, inactivate virus, attenuatedvirus or polysaccharides.

In another aspect, the disclosure provides immunogenic compositionscomprising one or more immunogens and the adjuvant compositionsdisclosed herein, wherein the immunogen is an immunogen-encodingpolynucleotide.

In another aspect, the disclosure provides immunogenic compositionscomprising one or more immunogens and the adjuvant compositionsdisclosed herein, wherein the immunogen-encoding polynucleotide is in apDNA vaccine.

In another aspect, the disclosure provides methods for immunizing avertebrate comprising administering to the vertebrate an immunogeniccomposition comprising a complex of one or more immunogen-encodingpolynucleotides and the adjuvant composition disclosed herein, in anamount sufficient to generate an immune response to the encodedimmunogen.

In another aspect, the disclosure provides methods for immunizing avertebrate, wherein the immunogenic composition further comprises one ormore co-lipids, including mono-, di-, and triacyl amphiphiles and mono-,di-, and trialkyl amphiphiles, such as fatty acids, monoacyl-,monoalkylglycerol, lysophospholipids and mono- and diacylglycerol,diacylphospholipids, and trialkyl or triacyl glycerol.

In another aspect, the disclosure provides methods for immunizing avertebrate, wherein the one or more co-lipids is DOPE, DPyPE, or DPyG.

In another aspect, the disclosure provides methods for providing amammal a prophylactic or therapeutic treatment associated with abacterial infection comprising: administering to the mammal animmunogenic composition comprising (a) one or more antigenicpolypeptides, immunogenic polypeptides or polysaccharides associatedwith the bacterial infection in an amount sufficient to generate animmune response to the one or more antigenic polypeptides, immunogenicpolypeptides or polysaccharides, and (b) the adjuvant compositiondisclosed herein. The antigenic polypeptides can be free in solution orderivatized with a hydrophobic moiety, such as an acyl chain, tofacilitate association with the adjuvant.

In another aspect, the disclosure provides methods for providing amammal a prophylactic or therapeutic treatment associated with a viralinfection comprising: administering to the mammal an immunogeniccomposition comprising (a) one or more antigenic polypeptides,immunogenic polypeptides, inactivate virus, attenuated virus orpolysaccharides associated with the viral infection in an amountsufficient to generate an immune response to the one or more antigenicpolypeptides, immunogenic polypeptides, inactivate virus, attenuatedvirus or polysaccharides, and (b) the adjuvant composition disclosedherein.

In another aspect, the disclosure provides methods for providing amammal a prophylactic or therapeutic treatment associated with anabnormal growth of a cell population comprising: administering to themammal an immunogenic composition comprising (a) one or more antigenicpolypeptides, immunogenic polypeptides or polysaccharides associatedwith the abnormal growth of the cell population in an amount sufficientto generate an immune response to the one or more antigenicpolypeptides, immunogenic polypeptides or polysaccharides, and (b) theadjuvant composition disclosed herein.

In another aspect, the disclosure provides pharmaceutical kitscomprising: (a) a container holding one or more antigenic polypeptides,immunogenic polypeptides, inactivate virus, attenuated virus orpolysaccharides, and (b) the adjuvant composition disclosed herein;wherein the one or more antigenic polypeptides, immunogenicpolypeptides, inactivate virus, attenuated virus or polysaccharides isprovided in a prophylactically or therapeutically effective amount totreat a vertebrate.

In another aspect, the disclosure provides compounds having formula II:

or an enantiomer, diastereomer, or a pharmaceutically acceptable salt orsolvate thereof, wherein:

R¹ and R² are each independently substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl,substituted or unsubstituted arylalkyl, substituted or unsubstitutedheteroarylalkyl, or substituted or unsubstituted carboxyalkyl, whereinR¹ and R² are each optionally independently substituted with 1 to 5 R¹⁶groups;

L₁ and L₂ are each independently a direct bond, O, NH, N(C₁-C₆ alkyl),or S(O)_(m), wherein m is an integer from 0 to 2;

n is an integer from 1 to 3;

Q′ is independently —N⁺Z₁- or —P⁺Z-;

Z₁ is independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl,substituted or unsubstituted arylalkyl, substituted or unsubstitutedheteroarylalkyl, substituted or unsubstituted carboxyalkyl, orsubstituted or unsubstituted —(CH₂)_(m)—R³ wherein m is an integer from1 to 6, and wherein Z₁ is optionally independently substituted with 1 to5 R¹⁶ groups;

T is substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl, substituted or unsubstitutedarylalkyl, substituted or unsubstituted heteroarylalkyl, or substitutedor unsubstituted carboxyalkyl; wherein T is optionally independentlysubstituted with 1 to 5 R¹⁶ groups; and

R³ is independently —OR⁴, —S(O)_(m)R⁵, —NR⁶R⁷, —N⁺R⁸R⁹R¹⁰, —PR¹¹R¹², or—P⁺R¹³R¹⁴R¹⁵;

R⁴ and R⁵ are each independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, or substituted or unsubstitutedcarboxyalkyl, wherein R⁴ and R⁵ are each optionally independentlysubstituted with 1 to 5 R¹⁶ groups;

R⁶ and R⁷ are each independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, substituted or unsubstitutedcarboxyalkyl, or R⁶ and R⁷, together with the N atom to which they areattached, form substituted or unsubstituted heteroaryl or substituted orunsubstituted heterocycloalkyl, wherein R⁶ and R⁷ are each optionallyindependently substituted with 1 to 5 R¹⁶ groups;

R⁸, R⁹ and R¹⁰ are each independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl,substituted or unsubstituted heteroarylalkyl, or substituted orunsubstituted carboxyalkyl, or R⁸ is as described, and R⁹ and R¹⁰,together with the N atom to which they are attached, form substituted orunsubstituted heteroaryl or substituted or unsubstitutedheterocycloalkyl, wherein R⁸, R⁹ and R¹⁰ are each optionallyindependently substituted with 1 to 5 R¹⁶ groups;

R¹¹, R¹² and R¹³ are each independently substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, substituted or unsubstitutedcarboxyalkyl, or R¹¹ is as described, and R¹² and R¹³, together with theP atom to which they are attached, form substituted or unsubstitutedheterocycloalkyl, wherein R¹¹, R¹² and R¹³ are each optionallyindependently substituted with 1 to 5 R¹⁶ groups;

R¹⁶ is hydrogen, halogen, nitro, cyano, hydroxyl, alkyl, cycloalkyl,perfluoroalkyl, heteroalkyl, heterocycloalkyl, aryl, arylalkyl,heteroaryl, heteroarylalkyl, —(CH₂)_(j)CN, —(CH₂)_(j)OR¹⁷,—(CH₂)_(j)C(O)R¹⁷, —(CH₂)_(j)C(O)OR¹⁷, —(CH₂)_(j)NR¹⁸R¹⁹,—(CH₂)_(j)C(O)NR¹⁸R¹⁹, —(CH₂)_(j)OC(O)NR¹⁸R¹⁹, —(CH₂)_(j)NR²C(O)R¹⁷,—(CH₂)_(j)NR²⁰C(O)OR¹⁷, —(CH₂)_(j)N²⁰C(O)NR¹⁸R¹⁹, —(CH₂)_(j)S(O)_(m)R²¹,—(CH₂)_(j)S(O)₂NR¹⁸R¹⁹, or —(CH₂)_(j)NR²⁰S(O)₂R²¹, wherein each j isindependently an integer from 0 to 6, and each m is independently aninteger from 0 to 2;

R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹ are each independently hydrogen, substitutedor unsubstituted alkyl, substituted or unsubstituted cycloalkyl,perfluoroalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted —O-aryl, substituted or unsubstitutedarylalkyl, substituted or unsubstituted heteroaryl, substituted orunsubstituted —O-heteroaryl, or substituted or unsubstitutedheteroarylalkyl, or

R¹⁷, R²⁰ and R²¹ are as described above, and R¹⁸ and R¹⁹, together withthe N atom to which they are attached, form substituted or unsubstitutedheteroaryl, or substituted or unsubstituted heterocycloalkyl; and one ormore counter ions.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II:

or an enantiomer, diastereomer, or a pharmaceutically acceptable salt orsolvate thereof, wherein:

R¹ and R² are each independently substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl,substituted or unsubstituted arylalkyl, substituted or unsubstitutedheteroarylalkyl, or substituted or unsubstituted carboxyalkyl, whereinR¹ and R² are each optionally independently substituted with 1 to 5 R¹⁶groups;

L₁ and L₂ are each independently a direct bond, O, NH, N(C₁-C₆ alkyl),or S(O)_(m), wherein m is an integer from 0 to 2;

n is an integer from 1 to 3;

Q′ is independently —N⁺Z₁- or —P⁺Z₁-;

Z₁ is independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl,substituted or unsubstituted arylalkyl, substituted or unsubstitutedheteroarylalkyl, substituted or unsubstituted carboxyalkyl, orsubstituted or unsubstituted —(CH₂)_(m)—R³ wherein m is an integer from1 to 6, and wherein Z₁ is optionally independently substituted with 1 to5 R¹⁶ groups;

T is substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl, substituted or unsubstitutedarylalkyl, substituted or unsubstituted heteroarylalkyl, or substitutedor unsubstituted carboxyalkyl; wherein T is optionally independentlysubstituted with 1 to 5 R¹⁶ groups; and

R³ is independently —OR⁴, —S(O)_(m)R⁵, —NR⁶R⁷, —N⁺R⁸R⁹R¹⁰, —PR¹¹R¹², or—P⁺R¹³R¹⁴R¹⁵;

R⁴ and R⁵ are each independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, or substituted or unsubstitutedcarboxyalkyl, wherein R⁴ and R⁵ are each optionally independentlysubstituted with 1 to 5 R¹⁶ groups;

R⁶ and R⁷ are each independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, substituted or unsubstitutedcarboxyalkyl, or R⁶ and R⁷, together with the N atom to which they areattached, form substituted or unsubstituted heteroaryl or substituted orunsubstituted heterocycloalkyl, wherein R⁶ and R⁷ are each optionallyindependently substituted with 1 to 5 R¹⁶ groups;

R⁸, R⁹ and R¹⁰ are each independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl,substituted or unsubstituted heteroarylalkyl, or substituted orunsubstituted carboxyalkyl, or R⁸ is as described, and R⁹ and R¹⁰,together with the N atom to which they are attached, form substituted orunsubstituted heteroaryl or substituted or unsubstitutedheterocycloalkyl, wherein R⁸, R⁹ and R¹⁰ are each optionallyindependently substituted with 1 to 5 R¹⁶ groups;

R¹¹, R¹² and R¹³ are each independently substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, substituted or unsubstitutedcarboxyalkyl, or R¹¹ is as described, and R¹² and R¹³, together with theP atom to which they are attached, form substituted or unsubstitutedheterocycloalkyl, wherein R¹¹, R¹² and R¹³ are each optionallyindependently substituted with 1 to 5 R¹⁶ groups;

R¹⁶ is hydrogen, halogen, nitro, cyano, hydroxyl, alkyl, cycloalkyl,perfluoroalkyl, heteroalkyl, heterocycloalkyl, aryl, arylalkyl,heteroaryl, heteroarylalkyl, —(CH₂)_(j)CN, —(CH₂)_(j)OR¹⁷,—(CH₂)_(j)C(O)R⁷, —(CH₂)_(j)C(O)OR⁷, —(CH₂)_(j)NR¹⁸R¹⁹,—(CH₂)_(j)C(O)NR¹⁸R¹⁹, —(CH₂)_(j)OC(O)NR¹⁸R¹⁹, —(CH₂)_(j)NR²⁰C(O)R¹⁷,—(CH₂)_(j)NR²⁰C(O)OR¹⁷, —(CH₂)_(j)N²⁰C(O)NR¹⁸R¹⁹, —(CH₂)_(j)S(O)_(m)R²¹,—(CH₂)_(j)S(O)₂NR¹⁸R¹⁹, or —(CH₂)_(j)NR²⁰S(O)₂R²¹, wherein each j isindependently an integer from 0 to 6, and each m is independently aninteger from 0 to 2;

R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹ are each independently hydrogen, substitutedor unsubstituted alkyl, substituted or unsubstituted cycloalkyl,perfluoroalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted —O-aryl, substituted or unsubstitutedarylalkyl, substituted or unsubstituted heteroaryl, substituted orunsubstituted —O-heteroaryl, or substituted or unsubstitutedheteroarylalkyl, or

R¹⁷, R²⁰ and R²¹ areas described above, and R¹⁸ and R¹⁹, together withthe N atom to which they are attached, form substituted or unsubstitutedheteroaryl, or substituted or unsubstituted heterocycloalkyl;

one or more counter ions; and

one or more co-lipids.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein:

R¹ and R² are each independently substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, or substituted or unsubstitutedalkynyl;

L₁ and L₂ are each independently O or NH;

n is 1; and

Q′ is independently -Z₁N⁺Z₂-.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein R¹ and R² are each independently substituted or unsubstituted(C₁-C₂₀)alkyl, substituted or unsubstituted (C₂-C₂₀)alkenyl, orsubstituted or unsubstituted (C₂-C₂₀)alkynyl.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein R¹ and R² are each independently:

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein:

Z₁ and Z₂ are each independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, or substituted or unsubstitutedcarboxyalkyl; and

T is substituted or unsubstituted alkyl.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein:

Z₁ and Z₂ are each independently (C₁-C₆)alkyl; and

T is independently (C₁-C₆)alkyl.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein:

Z₁ and Z₂ are each independently substituted or unsubstituted—(CH₂)_(m)—R³;

T is substituted or unsubstituted alkyl; and

R³ is independently —NR⁶R⁷ or —N⁺R⁸R⁹R¹⁰.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein:

T is independently (C₁-C₆)alkyl;

R³ is independently —NR⁶R⁷; and

R⁶ and R⁷ are each independently substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, or R⁶ and R⁷, together withthe N atom to which they are attached, form substituted or unsubstitutedheteroaryl or substituted or unsubstituted heterocycloalkyl.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein R⁶ and R⁷ are each independently substituted or unsubstituted(C₁-C₆)alkyl.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein R⁶ and R⁷, together with the N atom to which they are attached,form substituted or unsubstituted pyrrolyl, substituted or unsubstitutedimidazolyl, substituted or unsubstituted pyrrolidinyl, substituted orunsubstituted pyrazolinyl, substituted or unsubstituted piperidinyl,substituted or unsubstituted piperazinyl, substituted or unsubstitutedmorpholinyl, or substituted or unsubstituted thiomorpholinyl.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein:

T is independently (C₁-C₆)alkyl;

R³ is independently —N⁺R⁸R⁹R¹⁰; and

R⁸, R⁹ and R¹⁰ are each independently substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, or R⁸ is as described,and R⁹ and R¹⁰, together with the N atom to which they are attached,form substituted or unsubstituted heteroaryl or substituted orunsubstituted heterocycloalkyl.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein R⁸, R⁹ and R¹⁰ are each independently substituted orunsubstituted (C₁-C₆)alkyl.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein R⁹ and R¹⁰, together with the N atom to which they are attached,form substituted or unsubstituted pyrrolyl, substituted or unsubstitutedimidazolyl, substituted or unsubstituted pyrrolidinyl, substituted orunsubstituted pyrazolinyl, substituted or unsubstituted piperidinyl,substituted or unsubstituted piperazinyl, substituted or unsubstitutedmorpholinyl, or substituted or unsubstituted thiomorpholinyl.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein the counter ion is negatively charged.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein the negatively charged counter ion is F⁻ Cl⁻, Br⁻, I⁻, CH₃COO⁻,or CF₃COO⁻.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein the co-lipid is a neutral lipid.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein the neutral lipid is a phosphatidylethanolamine, and/or aphosphatidylcholine, and/or a mono-, di-, or trialkylglycerol, and/or amono-, di, or triacylglycerol, and/or a non-phospholipid zwitterionicco-lipid.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein the phosphatidylethanolamine is1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and/or1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE), and/or1,2-dimyristoyl-glycero-3-phosphoethanolamine (DMPE).

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein the phosphatidyl-ethanolamine is1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE).

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein the compound of formula II and the co-lipid ratio is from about9:1 to about 1:9.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein the compound of formula II and the co-lipid are in molar ratioof from about 4:1 to about 1:4.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein the compound of formula II and the co-lipid are in molar ratioof from about 2:1 to about 1:2.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein the compound of formula II and the co-lipid are in molar ratioof about 1:1.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein the compound of formula II and DPyPE are in molar ratio of fromabout 2:1 to about 1:2.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein the compound of formula II and DPyPE are in molar ratio of about1:1.

In another aspect, the disclosure provides adjuvant compositionscomprising a mixture of one or more cationic lipids having formula II,wherein the compound of formula II has formula:

In another aspect, the disclosure provides methods for immunizing avertebrate comprising administering into a tissue or cavity of thevertebrate an immunogenic composition comprising (a) one or moreantigenic polypeptides, immunogenic polypeptides, inactivate virus,attenuated virus or polysaccharides in an amount sufficient to generatean immune response to the one or more antigenic polypeptides,immunogenic polypeptides, inactivate virus, attenuated virus orpolysaccharides, and (b) the adjuvant compositions disclosed herein.

In another aspect, the disclosure provides methods for immunizing avertebrate comprising administering into a tissue or cavity of thevertebrate an immunogenic composition, wherein the immunogen-encodingpolynucleotide, upon incorporation into the cells of the vertebrate,produces an immunologically effective amount of an immunogen (e.g., animmunogenic protein).

In another aspect, the disclosure provides methods for immunizing avertebrate comprising administering into a tissue or cavity of thevertebrate an immunogenic composition, wherein the adjuvant compositionsdisclosed herein enhances the immune response of the vertebrate to theimmunogen.

In another aspect, the disclosure provides an immunogenic compositioncomprising one or more immunogens and the adjuvant compositionsdisclosed herein.

In another aspect, the disclosure provides an immunogenic compositioncomprising one or more immunogens and the adjuvant compositionsdisclosed herein, wherein the immunogen is one or more antigenicpolypeptides, immunogenic polypeptides, inactivate virus, attenuatedvirus or polysaccharides.

In another aspect, the disclosure provides an immunogenic compositioncomprising one or more immunogens and the adjuvant compositionsdisclosed herein, wherein the immunogen is an immunogen-encodingpolynucleotide.

In another aspect, the disclosure provides an immunogenic compositioncomprising one or more immunogens and the adjuvant compositionsdisclosed herein, wherein the immunogen-encoding polynucleotide is in apDNA vaccine.

In another aspect, the disclosure provides methods for immunizing avertebrate comprising administering to the vertebrate an immunogeniccomposition comprising a complex of one or more immunogen-encodingpolynucleotides and the adjuvant composition disclosed herein, in anamount sufficient to generate an immune response to the encodedimmunogen.

In another aspect, the disclosure provides methods for immunizing avertebrate comprising administering to the vertebrate an immunogeniccomposition comprising a complex of one or more immunogen-encodingpolynucleotides and the adjuvant composition disclosed herein, whereinthe immunogenic composition further comprises one or more co-lipids,including mono-, di-, and triacyl amphiphiles and mono-, di-, andtrialkyl amphiphiles, such as fatty acids, monoacyl-, monoalkylglycerol,lysophospholipids and mono- and diacylglycerol, diacylphospholipids, andtrialkyl or triacyl glycerol.

In another aspect, the disclosure provides methods for immunizing avertebrate comprising administering to the vertebrate an immunogeniccomposition comprising a complex of one or more immunogen-encodingpolynucleotides and the adjuvant composition disclosed herein, whereinthe one or more co-lipids is DOPE, DPyPE, and/or DPyG

In another aspect, the disclosure provides methods for providing amammal a prophylactic or therapeutic treatment associated with abacterial infection comprising: administering to the mammal animmunogenic composition comprising (a) one or more antigenicpolypeptides, immunogenic polypeptides or polysaccharides associatedwith the bacterial infection in an amount sufficient to generate animmune response to the one or more antigenic polypeptides, immunogenicpolypeptides or polysaccharides, and (b) the adjuvant compositiondisclosed herein.

In another aspect, the disclosure provides methods for providing amammal a prophylactic or therapeutic treatment associated with a viralinfection comprising: administering to the mammal an immunogeniccomposition comprising (a) one or more antigenic polypeptides,immunogenic polypeptides, inactivate virus, attenuated virus orpolysaccharides associated with the viral infection in an amountsufficient to generate an immune response to the one or more antigenicpolypeptides, immunogenic polypeptides, inactivate virus, attenuatedvirus or polysaccharides, and (b) the adjuvant composition disclosedherein.

In another aspect, the disclosure provides methods for providing amammal a prophylactic or therapeutic treatment associated with anabnormal growth of a cell population comprising: administering to themammal an immunogenic composition comprising (a) one or more antigenicpolypeptides, immunogenic polypeptides or polysaccharides associatedwith the abnormal growth of the cell population in an amount sufficientto generate an immune response to the one or more antigenicpolypeptides, immunogenic polypeptides or polysaccharides, and (b) theadjuvant composition disclosed herein.

In another aspect, the disclosure provides pharmaceutical kitscomprising: (a) a container holding one or more antigenic polypeptides,immunogenic polypeptides or polysaccharides, and (b) the adjuvantcomposition disclosed herein; wherein the one or more antigenicpolypeptides, immunogenic polypeptides or polysaccharides is provided ina prophylactically or therapeutically effective amount to treat avertebrate. The antigenic polypeptides can be free in solution orderivatized with a hydrophobic moiety, such as an acyl chain, tofacilitate association with the adjuvant.

The primary amine of GAP-DMORIE is chemically active and has thepotential to react with the ester of the helper lipid, or the phosphateof the DNA upon long term storage. This can result in inactivating thetransfection complex, resulting in a reduction in the shelf life of theproduct. Surprisingly, hypermethylating the amine eliminates thechemical reactivity but does not affect the adjuvant activity of thelipid. As is shown in the examples, dimethylation and trimethylation ofthe primary amine of GAP-DMORIE formulated with DPyPE in a 1:1 molarratio and complexed to DNA in a 4:1 mol/mol ratio of DNA phosphate tocationic lipid yielded equivalent protection in a mouse influenzachallenge model.

Primary amines are reactive nucleophiles. Long term storage of theseprimary amines in an aqueous media can produce chemical reactions thatare detrimental to a product, potentially impacting product stabilityresulting in reduced shelf life. Chemical reactivity can be controlledby adding substituent groups, such as methyl group (—CH₃), that reducechemical reactivity but maintain net charge. This later aspect of themodification is pertinent to development of cationic amphiphiles fordelivery of DNA. DNA can hydrogen bond and/or ionically bond withcationic amphiphiles. These two types of bonds dictate the interactionof DNA with cationic amphiphile based formulation for gene transfer.Hence, reducing the reactivity of a positively charged primary aminethrough derivatization with substituents groups does not eliminate theability of the phosphates from the DNA backbone to ion pair with thepositively charged quaternary amines.

Development of genetic vaccines requires the addition of the cationicamphiphiles to achieve sufficient expression to produce an immuneresponse. The cationic amphiphiles also possess the ability to activatethe immune system, thus increasing the potency of the vaccine. The exactmechanism for how these cationic amphiphiles accomplish immuneactivation is unknown. Also unknown are the chemical structurerequirements of the cationic amphiphiles to activate the immuneresponse. These unknowns limit predictability of chemical modificationson immune activation.

In this embodiment, conversion of a primary amine to a secondary amineor tertiary amine did not eliminate the adjuvant activity of thecationic amphiphile GAP-DMORIE in that both molecules formulated withDPyPE and complexed to flu antigen expression DNA plasmids resulted inequivalent survival of mice compared to the unmodified GAP-DMORIE whenexposed to a lethal dose of influenza virus. The trimethyl-GAP-DMORIEcan have an increased product shelf life compared to the unmodifiedGAP-DMORIE due to the reduction in reactivity of the primary amine.

As used herein, the term “co-lipid” refers to any hydrophobic materialthat can be combined with the cationic lipid component, e.g., thecompounds of formula I or II. The co-lipid of the present disclosure canbe amphipathic lipids, zwitterionic lipids, and non-ionic neutrallipids. Amphipathic lipids include phospholipids, e.g.,phosphatidylethanolamines and phosphatidylcholines. Non-phospholipidzwitterionic lipids include DPyRIE carboxylate or DMRIE carboxylate.Non-ionic neutral lipids include cholesterol and dialkylglycerols. Inone embodiment, phosphatidylethanolamines include but are not limited toDOPE and DPyPE. DPyPE comprises two phytanoyl substituents incorporatedinto the diacylphosphatidylethanolamine skeleton.

According to the present disclosure, the cationic lipid compounds offormula I or II, and co-lipids may be mixed or combined in a number ofways to produce a variety of adjuvant compositions of non-covalentlybonded macroscopic structures, e.g., liposomes, multilamellar vesicles,unilamellar vesicles, micelles, and simple films. These cationic lipidsand co-lipids can be mixed in a variety of molar ratios. In oneembodiment, the molar ratio for the compounds of formula I or II andco-lipid is from about 9:1 to about 1:9. In another embodiment, themolar ratio is from about 4:1 to about 1:4. In another embodiment, themolar ratio is from about 2:1 to about 1:2. In yet another embodiment,the molar ratio is about 1:1.

The cationic lipid compounds of formula I or II and co-lipids can bedissolved in a solvent to increase homogenity of the mixture. Suitablesolvents include chloroform. For example, the cationic lipid compound offormula I or II can be mixed with one or more co-lipids in chloroform,the mixture is subsequently evaporated under vacuum to form a dried thinlayer of film on the inner surface of a glass vessel, e.g., a rotovapround-bottomed flask. Such dried mixtures can be suspended in an aqueoussolvent where the amphipathic lipid component molecules self-assembleinto homogenous lipid vesicles. These lipid vesicles can subsequently beprocessed by any methods used in the art to have a selected meandiameter of uniform size prior to complexing with other entities, e.g.,pDNA. The sonication of a lipid solution is described in Felgner et al.,Proc. Natl. Acad. Sci. USA 84, 7413-7417 (1987) and in U.S. Pat. No.5,264,618, the disclosure of which is incorporated herein by reference.

The adjuvant compositions of the present disclosure may also includeadditives such as hydrophobic and amphiphilic additives. For example,the adjuvant composition can include sterols, fatty acids, gangliosides,glycolipids, lipopeptides, liposaccharides, neobees, niosomes,prostaglandins or sphingolipids. The amount of additives included in theadjuvant may be any including from about 0.1 mol % to about 99.9 mol %,from about 1 mol % to about 50 mol %, and from about 2 mol % to about 25mol %, relative to total amount of lipid. These additives can also beincluded in an immunogenic composition containing the adjuvantcomposition of the present disclosure.

The immunogenic composition of the present disclosure also includes anadjuvant composition as described above and an immunogen. An “immunogen”is meant to encompass any substance that when introduced into avertebrate, developing or developed, generates an immune response. Forexample, an immunogen may be a polypeptide with an amino acid sequencehaving one or more epitopes or combinations of epitopes, and animmunogen-encoding polynucleotide. An immunogen-encoding polynucleotidemay be part of a non-infectious and non-integrating linear expressioncassette, or a circular or linearized plasmid containing anon-infectious and non-integrating polynucleotide. A non-infectiouspolynucleotide is a polynucleotide that does not infect vertebrate cellswhile a non-integrating polynucleotide does not integrate into thegenome of vertebrate cells. A linearized plasmid is a plasmid that waspreviously circular but has been linearized, for example, by digestionwith a restriction endonuclease. Alternatively, the linear DNA can beproduced enzymatically by PCR. The immunogen-encoding polynucleotide maycomprise a sequence that directs the secretion of a polypeptide.Examples of polypeptides include, but are not limited to, those derivedfrom infectious agents such as bacteria, viruses, parasites, or fungi,allergens such as those from pet dander, plants, dust, and otherenvironmental sources, as well as certain self polypeptides, forexample, tumor-associated antigens or Amyloid Beta (Abeta) which is a39-43 amino acid peptide that is the main constituent of amyloid plaquesin the brains of Alzheimer's patients, or haptens, such as nicotine orcocaine derivatives used for drug abuse vaccines.

The immunogen-encoding polynucleotide is intended to encompass asingular “polynucleotide” as well as plural “polynucleotides,” andrefers to an isolated molecule or construct. The immunogen-encodingpolynucleotides include nucleotide sequences, nucleic acids, nucleicacid oligomers, messenger RNA (mRNA), DNA (e.g., pDNAs, derivatives ofpDNA, linear DNA or linear expression cassettes (LECs)), or fragments ofany of thereof. The immunogen-encoding polynucleotides may be providedin linear, circular, e.g., plasmid, or branched form as well asdouble-stranded or single-stranded form. The immunogen-encodingpolynucleotides may comprise a conventional phosphodiester bond or anon-conventional bond, e.g., an amide bond, such as found in peptidenucleic acids (PNA).

In addition, an “immunogen” is also meant to encompass anypoly-saccharide material, protein subunits or protein fragments andinactivated (protein-based) vaccines derived from whole virus particlesuseful in generating immune response.

The immunogenic composition of the present disclosure can also be usedto prevent or treat, i.e., cure, ameliorate, lessen the severity of, orprevent or reduce contagion of viral, bacterial, fungal, and parasiticinfectious diseases, as well as to treat allergies.

In addition, the immunogenic composition of the present disclosure canbe used to prevent or treat, i.e., cure, ameliorate, or lessen theseverity of cancer including, but not limited to, cancers of oral cavityand pharynx (i.e., tongue, mouth, pharynx), digestive system (i.e.,esophagus, stomach, small intestine, colon, rectum, anus, anal canal,anorectum, liver, gallbladder, pancreas), respiratory system (i.e.,larynx, lung), bones, joints, soft tissues (including heart), skin,melanoma, breast, reproductive organs (i.e., cervix, endometrium, ovary,vulva, vagina, prostate, testis, penis), urinary system (i.e., urinarybladder, kidney, ureter, and other urinary organs), eye, brain,endocrine system (i.e., thyroid and other endocrine), lymphoma (i.e.,hodgkin's disease, non-hodgkin's lynphoma), multiple myeloma, leukemia(i.e., acute lymphocytic leukemia, chronic lymphocytic leukemia, acutemyeloid leukemia, chronic myeloid leukemia).

Examples of viral immunogens include, but are not limited to, adenoviruspolypeptides, alphavirus polypeptides, calicivirus polypeptides, e.g., acalicivirus capsid antigen, coronavirus polypeptides, distemper viruspolypeptides, Ebola virus polypeptides, enterovirus polypeptides,flavivirus polypeptides, hepatitis virus (AE) polypeptides, e.g., ahepatitis B core or surface antigen, herpesvirus polypeptides, e.g., aherpes simplex virus or varicella zoster virus glycoprotein,immunodeficiency virus polypeptides, e.g., the human immunodeficiencyvirus envelope or protease, infectious peritonitis virus polypeptides,influenza virus polypeptides, e.g., an influenza A hemagglutinin,neuramimidase, or nucleoprotein, leukemia virus polypeptides, Marburgvirus polypeptides, orthomyxovirus polypeptides, papilloma viruspolypeptides, parainfluenza virus polypeptides, e.g., thehemagglutinin/neuramimidase, paramyxovirus polypeptides, parvoviruspolypeptides, pestivirus polypeptides, picoma virus polypeptides, e.g.,a poliovirus capsid polypeptide, pox virus polypeptides, e.g., avaccinia virus polypeptide, rabies virus polypeptides, e.g., a rabiesvirus glycoprotein G, reovirus polypeptides, retrovirus polypeptides,and rotavirus polypeptides.

Examples of bacterial immunogens include, but are not limited to,Actinomyces polypeptides, Bacillus polypeptides, Bacteroidespolypeptides, Bordetella polypeptides, Bartonella polypeptides, Borreliapolypeptides, e.g., B. burgdorferi OspA, Brucella polypeptides,Campylobacter polypeptides, Capnocytophaga polypeptides, Chlamydiapolypeptides, Clostridium polypeptides, Corynebacterium polypeptides,Coxiella polypeptides, Dermatophilus polypeptides, Enterococcuspolypeptides, Ehrlichia polypeptides, Escherichia polypeptides,Francisella polypeptides, Fusobacterium polypeptides, Haemobartonellapolypeptides, Haemophilus polypeptides, e.g., H. influenzae type b outermembrane protein, Helicobacter polypeptides, Klebsiella polypeptides,L-form bacteria polypeptides, Leptospira polypeptides, Listeriapolypeptides, Mycobacteria polypeptides, Mycoplasma polypeptides,Neisseria polypeptides, Neorickettsia polypeptides, Nocardiapolypeptides, Pasteurella polypeptides, Peptococcus polypeptides,Peptostreptococcus polypeptides, Pneumococcus polypeptides, Proteuspolypeptides, Pseudomonas polypeptides, Rickettsia polypeptides,Rochalimaea polypeptides, Salmonella polypeptides, Shigellapolypeptides, Staphylococcus polypeptides, Streptococcus polypeptides,e.g., S. pyogenes M proteins, Treponema polypeptides, and Yersiniapolypeptides, e.g., Y. pestis Fl and V antigens.

Examples of fungal immunogens include, but are not limited to, Absidiapolypeptides, Acremonium polypeptides, Alternaria polypeptides,Aspergillus polypeptides, Basidiobolus polypeptides, Bipolarispolypeptides, Blastomyces polypeptides, Candida polypeptides,Coccidioides polypeptides, Conidiobolus polypeptides, Cryptococcuspolypeptides, Curvalaria polypeptides, Epidermophyton polypeptides,Exophiala polypeptides, Geotrichum polypeptides, Histoplasmapolypeptides, Madurella polypeptides, Malassezia polypeptides,Microsporum polypeptides, Moniliella polypeptides, Mortierellapolypeptides, Mucor polypeptides, Paecilomyces polypeptides, Penicilliumpolypeptides, Phialemonium polypeptides, Phialophora polypeptides,Prototheca polypeptides, Pseudallescheria polypeptides,Pseudomicrodochium polypeptides, Pythium polypeptides, Rhinosporidiumpolypeptides, Rhizopus polypeptides, Scolecobasidium polypeptides,Sporothrix polypeptides, Stemphylium polypeptides, Trichophytonpolypeptides, Trichosporon polypeptides, and Xylohypha polypeptides.

Examples of protozoan parasite immunogens include, but are not limitedto, Babesia polypeptides, Balantidium polypeptides, Besnoitiapolypeptides, Cryptosporidium polypeptides, Eimeria polypeptides,Encephalitozoon polypeptides, Entamoeba polypeptides, Giardiapolypeptides, Hammondia polypeptides, Hepatozoon polypeptides, Isosporapolypeptides, Leishmania polypeptides, Microsporidia polypeptides,Neospora polypeptides, Nosema polypeptides, Pentatrichomonaspolypeptides, Plasmodium polypeptides, e.g., P. falciparumcircumsporozoite (PfCSP), sporozoite surface protein 2 (PfSSP2),carboxyl terminus of liver state antigen 1 (PfLSA1 c-term), and exportedprotein 1 (PfExp-1), Pneumocystis polypeptides, Sarcocystispolypeptides, Schistosoma polypeptides, Theileria polypeptides,Toxoplasma polypeptides, and Trypanosoma polypeptides.

Examples of helminth parasite immunogens include, but are not limitedto, Acanthocheilonema polypeptides, Aelurostrongylus polypeptides,Ancylostoma polypeptides, Angiostrongylus polypeptides, Ascarispolypeptides, Brugia polypeptides, Bunostomum polypeptides, Capillariapolypeptides, Chabertia polypeptides, Cooperia polypeptides, Crenosomapolypeptides, Dictyocaulus polypeptides, Dictyocaulus polypeptides,Dipetalonema polypeptides, Diphyllobothrium polypeptides, Diplydiumpolypeptides, Dirofilaria polypeptides, Dracunculus polypeptides,Enterobius polypeptides, Filaroides polypeptides, Haemonchuspolypeptides, Lagochilascaris polypeptides, Loa polypeptides, Mansonellapolypeptides, Muellerius polypeptides, Nanophyetus polypeptides, Necatorpolypeptides, Nematodirus polypeptides, Oesophagostomum polypeptides,Onchocerca polypeptides, Opisthorchis polypeptides, Ostertagiapolypeptides, Parafilaria polypeptides, Paragonimus polypeptides,Parascaris polypeptides, Physaloptera polypeptides, Protostrongyluspolypeptides, Setaria polypeptides, Spirocerca polypeptides Spirometrapolypeptides, Stephanofilaria polypeptides, Strongyloides polypeptides,Strongylus polypeptides, Thelazia polypeptides, Toxascaris polypeptides,Toxocara polypeptides, Trichinella polypeptides, Trichostrongyluspolypeptides, Trichuris polypeptides, Uncinaria polypeptides, andWuchereria polypeptides.

Examples of ectoparasite immunogens include, but are not limited to,polypeptides (including protective antigens as well as allergens) fromfleas; ticks, including hard ticks and soft ticks, flies, such asmidges, mosquitoes, sand flies, black flies, horse flies, horn flies,deer flies, tsetse flies, stable flies, myiasis-causing flies and bitinggnats; ants; spiders, lice; mites; and true bugs, such as bed bugs andkissing bugs.

Examples of tumor-associated immunogens include, but are not limited to,tumor-specific immunoglobulin variable regions, GM2, Tn, sTn,Thompson-Friedenreich antigen (TF), Globo H, Le(y), MUC1, MUC2, MUC3,MUC4, MUC5AC, MUC5B, MUC7, carcinoembryonic antigens, beta chain ofhuman chorionic gonadotropin (hCG beta), HER2/neu, PSMA, EGFRvIII, KSA,PSA, PSCA, GP100, MAGE 1, MAGE 2, TRP 1, TRP 2, tyrosinase, MART-1, PAP,CEA, BAGE, MAGE, RAGE, and related proteins.

Also included as immunogens of the present disclosure are fragments orvariants of the foregoing polypeptides, and any combination of theforegoing polypeptides. Furthermore, expressible nucleotide sequences ofeach of the foregoing polypeptides including fragments and/or variantsthereof are also included within the scope of the present disclosure.Additional polypeptides may be found, for example in “Foundations inMicrobiology,” Talaro, et al., eds., McGraw-Hill Companies (October1998), Fields, et al., “Virology,” 3rd ed., Lippincott-Raven (1996),“Biochemistry and Molecular Biology of Parasites;” Marr, et al., eds.,Academic Press (1995), and Deacon, J., “Modern Mycology,” BlackwellScience Inc (1997), which are incorporated herein by reference.

The form of immunogen-encoding polynucleotides depends in part on thedesired kinetics and duration of expression. When long-term delivery ofa protein encoded by a polynucleotide is desired, the preferred form isDNA. Alternatively, when short-term transgene protein delivery isdesired, the preferred form is mRNA, since mRNA can be rapidlytranslated into polypeptide, however RNA may be degraded more quicklythan DNA.

In one embodiment, the immunogen-encoding polynucleotide is RNA, e.g.,messenger RNA (mRNA). Methods for introducing RNA sequences intomammalian cells is described in U.S. Pat. No. 5,580,859, the disclosureof which is incorporated herein by reference. A viral alpha vector, anon-infectious vector useful for administering RNA, may be used tointroduce RNA into mammalian cells. Methods for the in vivo introductionof alpha viral vectors to mammalian tissues are described inAltman-Hamamdzic, S., et al., Gene Therapy 4, 815-822 (1997), thedisclosure of which is incorporated herein by reference.

In one embodiment, the immunogen-encoding polynucleotide is DNA. In thecase of DNA, a promoter is preferably operably linked to the nucleotidesequence encoding for the immunogen. The promoter can be a cell-specificpromoter that directs substantial transcription of the DNA only inpredetermined cells. Other transcription control elements, besides apromoter, can be included with the polynucleotide to directcell-specific transcription of the DNA. An operable linkage is a linkagein which a polynucleotide encoding for an immunogenic molecule isconnected to one or more regulatory sequences in such a way as to placeexpression of the immunogen under the influence or control of theregulatory sequence(s). Two DNA sequences (such as a coding sequence anda promoter region sequence linked to the 5′ end of the coding sequence)are operably linked if induction of promoter function results in thetranscription of mRNA encoding for the desired immunogen and if thenature of the linkage between the two DNA sequences does not (1) resultin the introduction of a frame-shift mutation, (2) interfere with theability of the expression regulatory sequences to direct the expressionof the immunogen, or (3) interfere with the ability of the DNA templateto be transcribed. Thus, a promoter region would be operably linked to aDNA sequence if the promoter was capable of effecting transcription ofthat DNA sequence.

The immunogen-encoding polynucleotide, e.g., pDNA, mRNA, polynucleotideor nucleic acid oligomer can be solubilized in any of various buffersprior to mixing or complexing with the adjuvant components, e.g.,cationic lipids and co-lipids. Suitable buffers include phosphatebuffered saline (PBS), normal saline, Tris buffer, and sodium phosphate.Insoluble polynucleotides can be solubilized in a weak acid or weakbase, and then diluted to the desired volume with a buffer. The pH ofthe buffer may be adjusted as appropriate. In addition, apharmaceutically acceptable additive can be used to provide anappropriate osmolarity. Such additives are within the purview of oneskilled in the art.

According to the present disclosure, the immunogen-encodingpolynucleotides can be complexed with the adjuvant compositions of thepresent disclosure by any means known in the art, e.g., by mixing a pDNAsolution and a solution of cationic lipid/co-lipid liposomes. In oneembodiment, the concentration of each of the constituent solutions isadjusted prior to mixing such that the desired final pDNA/cationiclipid:co-lipid ratio and the desired pDNA final concentration will beobtained upon mixing the two solutions. For example, if the desiredfinal solution is to be physiological saline (0.9% weight/volume), bothpDNA and cationic lipid:co-lipid liposomes are prepared in 0.9% salineand then simply mixed to produce the desired complex. The cationiclipid:co-lipid liposomes can be prepared by any means known in the art.For example, one can hydrate a thin film of the cationic lipid compoundof formula I or II and co-lipid mixture in an appropriate volume ofaqueous solvent by vortex mixing at ambient temperatures for about 1minute. Preparation of a thin film of the cationic lipid and co-lipidmixture is known to a skilled artisan and can be prepared by anysuitable techniques. For example, one can mix chloroform solutions ofthe individual components to generate an equimolar solute ratio andsubsequently aliquot a desired volume of the solutions into a suitablecontainer where the solvent can be removed by evaporation, e.g., firstwith a stream of dry, inert gas such as argon and then by high vacuumtreatment.

The immunogenic composition of the present disclosure can be used toimmunize a vertebrate. The term “vertebrate” is intended to encompass asingular “vertebrate” as well as plural “vertebrates”, and comprisesdeveloping and developed mammalian and avian species, as well as fish.The method for immunizing a vertebrate includes administering to thevertebrate an immunogenic composition of the present disclosure in anamount sufficient to generate an immune response to the immunogen.

The immunogenic compositions of the present disclosure may beadministered according to any of various methods known in the art. Forexample, U.S. Pat. No. 5,676,954 reports on the injection of geneticmaterial, complexed with cationic lipid carriers, into mice. Also, U.S.Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and PCTinternational patent application PCT/US94/06069 (WO 94/29469), thedisclosures of which are incorporated herein by reference, providemethods for delivering DNA-cationic lipid complexes to mammals.

Specifically, the immunogenic compositions of the present disclosure maybe administered to any tissue of a vertebrate, including, but notlimited to, muscle, skin, sub-cutaneous space, brain, lung, liver,spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage,mucosal tissue, pancreas, kidney, gall bladder, stomach, intestine,testis, ovary, uterus, vaginal tissue, rectum, nervous system, eye,gland, tongue and connective tissue. The immunogenic compositions of thedisclosure may also be administered to a body cavity, including, but notlimited to, the lung, mouth, nasal cavity, stomach, peritoneum,intestine, heart chamber, vein, artery, capillary, lymphatic, uterus,vagina, rectum, and ocular cavity.

The immunogenic compositions of the present disclosure may beadministered by intramuscular (i.m.), intranasal (i.n.), topical,intradermal (i.d.), epidermal, tansdermal, transcutaneous orsubcutaneous (s.c.) routes. Other suitable routes of administrationinclude transdermal, intranasal, tattooing, inhalation, intratracheal,transmucosal (i.e., across a mucous membrane), intra-cavity (e.g., oral,vaginal, or rectal), intraocular, vaginal, rectal, intraperitoneal,intraintestinal and intravenous (i.v.) administration.

Any mode of administration can be used so long as the administrationresults in desired immune response. Administration means of the presentdisclosure include, but not limited to, microneedles, sonoporation,ionotophoresis, transdermal/transcutaneous delivery, microseeding(tattoo devises), needle injection, electroporation, catheter infusion,biolistic injectors, particle accelerators (i.e., “gene guns”) orpneumatic “needleless” injectors—for example, Med-E-Jet (Vahlsing, H.,et al., J. Immunol. Methods 171, 11-22 (1994)), Pigjet (Schrijver, R.,et al., Vaccine 15, 1908-1916 (1997)), Biojector (Davis, H., et al.,Vaccine 12, 1503-1509 (1994); Gramzinski, R., et al., Mol. Med. 4,109-118 (1998)), AdvantaJet, Medijector, gelfoam sponge depots, othercommercially available depot materials (e.g., hydrogels), osmotic pumps(e.g., Alza minipumps), oral or suppositorial solid (tablet or pill)pharmaceutical formulations, spray/inhalers, topical skin creams,patches, and decanting, use of polynucleotide coated suture (Qin et al.,Life Sciences 65, 2193-2203 (1999)) or topical applications duringsurgery. In an embodiment, the mode of administration is intramuscularneedle-based injection or intranasal application as an aqueous solution.

Determining an effective amount of an immunogenic composition dependsupon a number of factors including, for example, the chemical structureand biological activity of the substance, the age and weight of thesubject, and the route of administration. The precise amount, number ofdoses, and timing of doses can be readily determined by those skilled inthe art.

In certain embodiments, the immunogenic composition is administered as apharmaceutical composition. Such a pharmaceutical composition can beformulated according to known methods, whereby the substance to bedelivered is combined with a pharmaceutically acceptable carriervehicle. Suitable vehicles and their preparation are described, forexample, in Remington's Pharmaceutical Sciences, 16^(th) Edition, A.Osol, ed., Mack Publishing Co., Easton, Pa. (1980), and Remington'sPharmaceutical Sciences, 19^(th) Edition, A. R. Gennaro, ed., MackPublishing Co., Easton, Pa. (1995). The pharmaceutical composition canbe formulated as an emulsion, gel, solution, suspension, lyophilizedform, or any other form known in the art. In addition, thepharmaceutical composition can also contain pharmaceutically acceptableadditives including, for example, diluents, binders, stabilizers, andpreservatives. Administration of pharmaceutically acceptable salts ofthe polynucleotide constructs described herein is preferred. Such saltscan be prepared from pharmaceutically acceptable non-toxic basesincluding organic bases and inorganic bases. Salts derived frominorganic bases include sodium, potassium, lithium, ammonium, calcium,magnesium, and the like. Salts derived from pharmaceutically acceptableorganic non-toxic bases include salts of primary, secondary, andtertiary amines, basic amino acids, and the like.

For aqueous pharmaceutical compositions used in vivo, use of sterilepyrogen-free water is preferred. Such formulations will contain aneffective amount of the immunogenic composition together with a suitableamount of vehicle in order to prepare pharmaceutically acceptablecompositions suitable for administration to a vertebrate.

The present disclosure also provides kits for use in delivering apolypeptide to a vertebrate. Each kit includes a container holding 1 ngto 30 mg of an immunogen-encoding polynucleotide which operably encodesan immunogen within vertebrate cells in vivo. Furthermore, each kitincludes, in the same or in a different container, an adjuvantcomposition comprising a cationic lipid compound of formula I or II anda co-lipid. Any of components of the pharmaceutical kits can be providedin a single container or in multiple containers. In one embodiment, thekit includes from about 1 ng to about 30 mg of a immunogen-encodingpolynucleotide. In another embodiment, the kit includes from about 100ng to about 10 mg of a immunogen-encoding polynucleotide.

Any suitable container or containers may be used with pharmaceuticalkits. Examples of containers include, but are not limited to, glasscontainers, plastic containers, or strips of plastic or paper.

Each of the pharmaceutical kits may further comprise an administrationmeans. Means for administration include, but are not limited to syringesand needles, catheters, biolistic injectors, particle accelerators,i.e., “gene guns,” pneumatic “needleless” injectors, gelfoam spongedepots, other commercially available depot materials, e.g., hydrojels,osmotic pumps, and decanting or topical applications during surgery(topical patch-such as those used for nicotine delivery-passive oractive administration). Each of the pharmaceutical kits may furthercomprise sutures, e.g., coated with the immunogenic composition (Qin etal., Life Sciences (1999) 65:2193-2203).

The kit can further comprise an instruction sheet for administration ofthe composition to a vertebrate. The polynucleotide components of thepharmaceutical composition are preferably provided as a liquid solutionor they may be provided in lyophilized form as a dried powder or a cake.If the polynucleotide is provided in lyophilized form, the dried powderor cake may also include any salts, entry enhancing agents, transfectionfacilitating agents, and additives of the pharmaceutical composition indried form. Such a kit may further comprise a container with an exactamount of sterile pyrogen-free water, for precise reconstitution of thelyophilized components of the pharmaceutical composition.

The container in which the pharmaceutical composition is packaged priorto use can comprise a hermetically sealed container enclosing an amountof the lyophilized formulation or a solution containing the formulationsuitable for a pharmaceutically effective dose thereof, or multiples ofan effective dose. The pharmaceutical composition is packaged in asterile container, and the hermetically sealed container is designed topreserve sterility of the pharmaceutical formulation until use.Optionally, the container can be associated with administration meansand/or instruction for use.

Pharmaceutical Compositions and Administration

In another aspect, the present disclosure relates to pharmaceuticalcompositions including the cationic lipid compound of formula I or II,and a co-lipid in a mixture with a pharmaceutically acceptableexcipient. One of skill in the art will recognize that thepharmaceutical compositions include the pharmaceutically acceptablesalts of the compounds described above.

In therapeutic and/or diagnostic applications, the compounds of thedisclosure can be formulated for a variety of modes of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remington: The Science andPractice of Pharmacy (20^(th) ed.) Lippincott, Williams & Wilkins(2000).

The compounds according to the disclosure are effective over a widedosage range. For example, in the treatment of adult humans, dosagesfrom 0.01 to 10,000 mg, from 0.5 to 1000 mg, from 1 to 500 mg per day,and from 5 to 100 mg per day are examples of dosages that may be used.The exact dosage will depend upon the route of administration, the formin which the compound is administered, the subject to be treated, thebody weight of the subject to be treated, and the preference andexperience of the attending physician.

Pharmaceutically acceptable salts are generally well known to those ofordinary skill in the art, and may include, by way of example but notlimitation, acetate, benzenesulfonate, besylate, benzoate, bicarbonate,bitartrate, bromide, calcium edetate, carnsylate, carbonate, citrate,edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate,napsylate, nitrate, pamoate (embonate), pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate,subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Otherpharmaceutically acceptable salts may be found in, for example,Remington: The Science and Practice of Pharmacy (20^(th) ed.)Lippincott, Williams & Wilkins (2000). Preferred pharmaceuticallyacceptable salts include, for example, acetate, benzoate, bromide,carbonate, citrate, gluconate, hydrobromide, hydrochloride, maleate,mesylate, napsylate, pamoate (embonate), phosphate, salicylate,succinate, sulfate, or tartrate.

Depending on the specific conditions being treated, such agents may beformulated into liquid or solid dosage forms and administeredsystemically or locally. The agents may be delivered, for example, in atimed- or sustained-low release form as is known to those skilled in theart. Techniques for formulation and administration may be found inRemington: The Science and Practice of Pharmacy (20^(th) ed.)Lippincott, Williams & Wilkins (2000). Suitable routes may include oral,buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal,transmucosal, nasal or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intra-articullar, intra-sternal, intra-synovial, intra-hepatic,intralesional, intracranial, intraperitoneal, intranasal, or intraocularinjections or other modes of delivery.

For injection, the agents of the disclosure may be formulated anddiluted in aqueous solutions, such as in physiologically compatiblebuffers such as Hank's solution, Ringer's solution, or physiologicalsaline buffer. For such transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

Use of pharmaceutically acceptable inert carriers to formulate thecompounds herein disclosed for the practice of the disclosure intodosages suitable for systemic administration is within the scope of thedisclosure. With proper choice of carrier and suitable manufacturingpractice, the compositions of the present disclosure, in particular,those formulated as solutions, may be administered parenterally, such asby intravenous injection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe disclosure to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated.

For nasal or inhalation delivery, the agents of the disclosure may alsobe formulated by methods known to those of skill in the art, and mayinclude, for example, but not limited to, examples of solubilizing,diluting, or dispersing substances such as, saline, preservatives, suchas benzyl alcohol, absorption promoters, and fluorocarbons.

Pharmaceutical compositions suitable for use in the present disclosureinclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipients, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations, for example, maize starch, wheat starch, rice starch,potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC),and/or polyvinylpyrrolidone (PVP: povidone). If desired, disintegratingagents may be added, such as the cross-linked polyvinylpyrrolidone,agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol (PEG), and/or titanium dioxide, lacquer solutions, and suitableorganic solvents or solvent mixtures. Dye-stuffs or pigments may beadded to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin, and a plasticizer, such as glycerol or sorbitdl. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols (PEGs). In addition, stabilizers may be added.

Depending upon the particular condition, or disease state, to be treatedor prevented, additional therapeutic agents, which are normallyadministered to treat or prevent that condition, may be administeredtogether with the inhibitors of this disclosure. For example,chemotherapeutic agents or other anti-proliferative agents may becombined with the inhibitors of this disclosure to treat proliferativediseases and cancer. Examples of known chemotherapeutic agents include,but are not limited to, adriamycin, dexamethasone, vincristine,cyclophosphamide, fluorouracil, topotecan, taxol, interferons, andplatinum derivatives.

Other examples of agents the inhibitors of this disclosure may also becombined with include, without limitation, anti-inflammatory agents suchas corticosteroids, TNF blockers, IL-1 R^(A), azathioprine,cyclophosphamide, and sulfasalazine; immunomodulatory andimmunosuppressive agents such as cyclosporin, tacrolimus, rapamycin,mycophenolate mofetil, interferons, corticosteroids, cyclophophamide,azathibprine, and sulfasalazine; neurotrophic factors such asacetylcholinesterase inhibitors, MAO inhibitors, interferons,anti-convulsants, ion channel blockers, riluzole, and anti-Parkinsonianagents; agents for treating cardiovascular disease such asbeta-blockers, ACE inhibitors, diuretics, nitrates, calcium channelblockers, and statins; agents for treating liver disease such ascorticosteroids, cholestyramine, interferons, and anti-viral agents;agents for treating blood disorders such as corticosteroids,anti-leukemic agents, and growth factors; agents for treating diabetessuch as insulin, insulin analogues, alpha glucosidase inhibitors,biguanides, and insulin sensitizers; and agents for treatingimmunodeficiency disorders such as gamma globulin.

These additional agents may be administered separately, as part of amultiple dosage regimen, from the inhibitor-containing composition.Alternatively, these agents may be part of a single dosage form, mixedtogether with the inhibitor in a single composition.

Exemplary Syntheses

The compounds of the disclosure are synthesized by an appropriatecombination of generally well known synthetic methods. Techniques usefulin synthesizing the compounds of the disclosure are both readilyapparent and accessible to those of skill in the relevant art. Thediscussion below is offered to illustrate certain of the diverse methodsavailable for use in assembling the compounds of the disclosure.However, the discussion is not intended to define the scope of reactionsor reaction sequences that are useful in preparing the compounds of thepresent disclosure. The compounds of this disclosure may be made by theprocedures and techniques disclosed in the Examples section below, aswell as by known organic synthesis techniques.

The compounds of the present disclosure may be synthesized using one ormore protecting groups generally known in the art of chemical synthesis.The term “protecting group” refers to chemical moieties that block someor all reactive moieties of a compound and prevent such moieties fromparticipating in chemical reactions until the protective group isremoved, for example, those moieties listed and described in Greene, etal., Protective Groups in Organic Synthesis, 3rd ed. John Wiley & Sons(1999). It may be advantageous, where different protecting groups areemployed, that each (different) protective group be removable by adifferent means. Protective groups that are cleaved under totallydisparate reaction conditions allow differential removal of suchprotecting groups. For example, protective groups can be removed byacid, base, and hydrogenolysis. Groups such as trityl, dimethoxytrityl,acetal and t-butyldimethylsilyl are acid labile and may be used toprotect carboxy and hydroxy reactive moieties in the presence of aminogroups protected with Cbz groups, which are removable by hydrogenolysis,and Fmoc groups, which are base labile. Carboxylic acid and hydroxyreactive moieties may be blocked with base labile groups such as,without limitation, methyl, ethyl, and acetyl in the presence of aminesblocked with acid labile groups such as t-butyl carbamate or withcarbamates that are both acid and base stable but hydrolyticallyremovable.

Carboxylic acid and hydroxy reactive moieties may also be blocked withhydrolytically removable protective groups such as the benzyl group,while amine groups capable of hydrogen bonding with acids may be blockedwith base labile groups such as Fmoc. Carboxylic acid reactive moietiesmay be blocked with oxidatively-removable protective groups such as2,4-dimethoxybenzyl, while co-existing amino groups may be blocked withfluoride labile silyl carbamates.

Allyl blocking groups are useful in the presence of acid- andbase-protecting groups since the former are stable and can besubsequently removed by metal or pi-acid catalysts. For example, anallyl-blocked carboxylic acid can be deprotected with apalladium(0)-catalyzed reaction in the presence of acid labile t-butylcarbamate or base-labile acetate amine protecting groups. Yet anotherform of protecting group is a resin to which a compound or intermediatemay be attached. As long as the residue is attached to the resin, thatfunctional group is blocked and cannot react. Once released from theresin, the functional group is available to react.

Typical blocking or protecting groups include, for example:

The present disclosure is not to be limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe disclosure. Indeed, various modifications of the disclosure inaddition to those described herein will become apparent to those havingskill in the art from the foregoing description. Such modifications areintended to fall within the scope of the disclosure. Moreover, any oneor more features of any embodiment of the disclosure may be combinedwith any one or more other features of any other embodiment of thedisclosure, without departing from the scope of the disclosure.References cited throughout this application are examples of the levelof skill in the art and are hereby incorporated by reference herein intheir entirety for all purposes, whether previously specificallyincorporated or not.

EXAMPLES A. Synthesis of Compounds of the Present Invention

The following examples are offered to illustrate, but not to limit theclaimed disclosure. The preparation of embodiments of the presentdisclosure is described in the following examples. Those of ordinaryskill in the art will understand that the chemical reactions andsynthesis methods provided may be modified to prepare many of the othercompounds of the present disclosure. Where compounds of the presentdisclosure have not been exemplified, those of ordinary skill in the artwill recognize that these compounds may be prepared by modifyingsynthesis methods presented herein, and by using synthesis methods knownin the art.

Example I Synthesis of(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(myristoleyloxy)-1-propanaminiumBromide (GAP-DMORIE, Formula I)

Racemic 1-dimethylamino-2,3-propanediol (0.96 g; Janssen Chimica) wasconverted to the disodium salt in situ by treatment with sodium hydride(60% in oil, 0.8 g) in tetrahydrofuran (70 mL). Condensation withmyristoleyl methane sulfonate (5.3 g; NuChek Prep) afforded crude(±)-N,N-dimethyl-(2,3-bis(myristoleyloxy))propylamine (DMOP-DMA). Thismaterial was purified to homogeneity by silica gel chromatographyemploying a step gradient of ether in hexane (from 10% to 50%), andfinally neat ether, as the eluents. DMOP-DMA (2.4 g) was then treatedwith N-(3-bromopropyl)phthalimide (2.5 g) in dimethylformamide (15 mL)at elevated temperature (85° C., overnight) to effect quaternization ofthe amine. Removal of the dimethylformamide in vacuo followed by silicagel chromatography using a step gradient of methanol/chloroform as theeluent afforded TLC homogenous material. Deprotection of the primaryamine was accomplished by treatment of the phthalimide (2.1 g) withanhydrous hydrazine (1.7 mL) in anhydrous ethanol (40 mL) and propargylalcohol (10 eq.). Filtration, evaporative removal of the solvent, basicextraction (0.1 M NaOH), alumina chromatography, and washing with 1 MNaBr afforded the pure product.

Example II Synthesis of(±)-N-(3‘-N’-propyl-N′,N′,N′-trimethylammonium)-N,N-dimethyl-2,3-bis(myristoleyloxy)-1-propanaminiumdibromide (Trimethyl-GAP-DMORIE, Formula I)

GAP-DMORIE (0.64 g) was treated with 1 M NaOH (4.2 mL) and CH₃I (3 mL)in methanol (20 mL) at 0° C. to room temperature overnight to effectquaternization of the primary amine. The solvent was removed in vacuo,and the residue was taken up in CHCl₃ and washed with water. The organiclayer was dried over Na₂SO₄, filtered, and concentrated. The residue waspurified by chromatography on neutral alumina using 78/20/2/0.5CHCl₃/MeOH/H₂O/15% NH₄OH as the eluent. The appropriate fractions werepooled and concentrated. The residue was taken up in CHCl₃, washed with1 M NaBr, dried (Na₂SO₄), and concentrated to afford the pure product.

Example III Synthesis of(±)-N-(3′-N″-propyl-N′,N′,N′-trimethylammonium)-N,N-dimethyl-2,3-bis(lauryloxy)-1-propanaminiumdibromide Trimethyl-GAP-DLRIE, Formula I)

DLP-DMA (0.25 g, prepared analogously to DMOP-DMA above) and3-bromopropyl-trimethylammonium bromide (430 mg) were combined in DMF (5mL) and stirred overnight at 75° C. under argon atmosphere to effectquaternization of the tertiary amine. The solvent was removed bydistillation in vacuo, and the dry residue was purified by silica gelchromatography using 70/30 CHCl₃/MeOH as the eluent. The appropriatefractions were pooled and concentrated to afford the pure product.

Example IV Synthesis of(±)-N,N-Bis(3′-N′-propyl-N′,N′,N′-trimethylammonium)-N-methyl-2,3-bis(tetradecyloxy)-1-propanaminiumtribromide (Hexamethyl-BPA-DMRIE, Formula I)

Bis(propylamino)-DMRIE (0.60 g) was treated with 9 M NaOH (0.7 mL) andCH₃I (3 mL) in methanol (20 mL) at 0° C. to room temperature overnightto effect quaternization of both primary amines. The solvent was removedin vacuo, and the residue was purified by chromatography on neutralalumina using 78/20/2/0.5 CHCl₃/MeOH/H₂O/15% NH₄OH as the eluent. Theappropriate fractions were pooled and concentrated. The residue wastaken up in CHCl₃, washed with 1 M NaBr, dried (Na₂SO₄), andconcentrated to afford the pure product.

Example V Synthesis of(±)-N-(3-dimethylaminopropyl)-N,N-dimethyl-2,3-bis(myristoleyloxy)-1-propanaminiumBromide (Dimethyl-GAP-DMORIE, Formula I)

DMOP-DMA (2.2 g, prepared as above) and 1,3-dibromopropane (1 mL) werecombined in DMF (20 mL) and stirred overnight at 85° C. under argonatmosphere to effect quaternization of the tertiary amine. The solventwas removed by distillation in vacuo, and the dry residue was purifiedby silica gel chromatography using 85/15 CHCl₃/MeOH as the eluent. Theappropriate fractions were pooled and concentrated to afford the pure3-bromopropyl-DMORIE intermediate. This intermediate (0.7 g) wasrefluxed with dimethylamine (10 mL, 2.0 M in MeOH) in methanol (10 mL)overnight. The solvent was removed in vacuo and the crude product waschromatographed on silica gel using 78/20/2 CHCl₃/MeOH/H₂O as theeluent. The appropriate fractions were pooled and concentrated to affordthe pure product.

Example VI Synthesis ofN-(3-Aminopropyl)-N-methyl-4,4-bis(oleyloxy)-piperidinium Bromide(PA-DOMe-Pipd, Formula II)

A solution of N-methyl-4-piperidone (5.03 g), oleyl alcohol (26.3 g),and p-TsOH (9.32 g) in benzene was refluxed for one hour with stirring,and the water removed by a Dean-Stark trap. After cooling to roomtemperature, the solution was diluted with ether and washed sequentiallywith 1 M NaOH and brine. The organic layer was dried (Na₂SO₄) andconcentrated to afford the crude intermediate as an oil. This materialwas purified by silica gel chromatography employing a step gradient ofCHCl₃ and 90/10 CHCl₃/MeOH as the eluents, affording 16.3 g pure product(DOMe-pipd). DOMe-pipd (7.8 g) was then combined withN-(3-bromopropyl)phthalimide (4.0 g) in dimethylformamide (20 mL) atelevated temperature (85° C., overnight) to effect quaternization of thepiperidine ring. Distillation of the dimethylformamide in vacuo followedby silica gel chromatography employing a step gradient of 95/5CHCl₃/MeOH and 90/10 CHCl₃/MeOH as the eluents, afforded 6.0 g pureproduct (γ-Phth-DOMe-pipd). Deprotection of the primary amine wasaccomplished by treatment of the phthalimide with anhydrous hydrazine(4.1 mL) in anhydrous ethanol (100 mL) and propargyl alcohol (10 eq.).Filtration, evaporative removal of the solvent, basic extraction (0.1 MNaOH), alumina chromatography, and 1 M NaBr wash afforded the titlecompound.

The following example demonstrates the surprising finding that thecationic lipid compounds of formula I and/or II:co-lipid complexed withan antigen-encoding pDNA can enhance subsequent immune response comparedto presently known nucleic acid immunization methods when administeredinto murine or rabbit tissues.

Example VII Formulation and In Vivo Study of Trimethyl-GAP-DMORIE/DPyPEin the Murine Flu Challenge Model

Trimethyl-GAP-DMORIE/DPyPE (1:1 molar ratio, 2.25 mg total lipid) drylipid films were prepared and suspended in 0.9% saline (1 mL). 0.7 mL ofthis suspension was added slowly to 0.7 mL of a cocktail containingplasmids encoding for NP and M2 influenza viral surface proteins in 20mM PBS (2 mg/mL total plasmid) to afford the final pDNA/lipidformulation at 1 mg/mL (10 mM PBS, pDNA/lipid ratio at 4:1). Theformulation was then diluted further with PBS to a concentration of 0.05ug/uL prior to injection. Each mouse received 100 uL of pDNA/lipidformulation (0.05 ug/uL), injected bilaterally into the rectus femorismuscle at 0 and 3 weeks. Mice were given a lethal challenge of flu virusat week 6, and survival was measured three weeks later. Mice treatedwith either trimethyl- or dimethyl-GAP-DMORIE/DPyPE/pDNA formulationsall exhibited high survival rates (80-100% protection).

B. Delivery Methods of Formulated DNA

The following examples demonstrate the surprising finding that the modeof delivering formulated DNA impacts both the humoral as well ascellular immune responses. All scientific and technical terms have themeanings as understood by one with ordinary skill in the art. Thespecific examples which follow illustrate the methods in which thecompositions of the present invention may be administered and are not tobe construed as limiting the invention in sphere or scope.

Materials and Methods Reagents

The nonionic tri-block copolymer (poloxamer) CRL1005 was obtained fromCytRx Corporation (Los Angeles, Calif.). The cationic surfactantBenzalkonium chloride 50% solution NF (BAK, BTC 50® NF) was obtainedfrom Stepan Company (Northfield, Ill.). Vaxfectin® was made inaccordance with U.S. Pat. Nos. 6,586,405 and 7,105,574 which areincorporated herein by reference.

Plasmid Constructs

Influenza antigen plasmids VR4752 (HI HA; negative control plasmid usedin influenza challenge studies), VR4750 (H3 HA; positive control plasmidused in influenza challenge studies), VR4759 (encoding M2) and VR4762(encoding NP) were constructed by inserting the influenza Hi HA, H3 HA,M2, or NP gene open reading frame sequence into expression plasmidVR10551 containing the human CMV immediate early 1 promoter/enhancer andintron A, modified rabbit β-globin terminator and kanamycin resistancegene as previously described (Jimenez, G. S., et al., Hum Vaccine, 3(5),157-64 (2007). Plasmid VCL6365 encoding a secreted form of human CMV gBantigen lacking the transmembrane and cytoplasmic domains wasconstructed as previously described (Selinsky, C., et al., Hum Vaccine,1(1), 16-23 (2005).

Preparation of Adjuvant and Immunogenic Compositions Cytofectin:Co-lipidAdjuvant Formulations

Cytofectin:co-lipid adjuvant formulations were prepared as previouslydescribed (Hartikka, J., et al., Vaccine, 19(15-16), 1911-23 (2001);Jimenez, G. S., et al., Hum Vaccine, 3(5), 157-64 (2007). DMRIE:DOPE(DM:DP) consists of a 1:1 molar mixture of DMRIE and DOPE, andVaxfectin® consists of a 1:1 molar mixture of VC1052 and DPyPE. BothDM:DP and Vaxfectin® were prepared as dried lipid films by mixingequimolar chloroform solutions of DM and DP or VC1052 and DPyPE,respectively. The chloroform was evaporated under a stream of nitrogenand then the vials were placed under vacuum overnight. For DM:DP, thelipid film was reconstituted with 1 ml of PBS, followed by vortexing for5 minutes, while the Vaxfectin® lipid film was reconstituted with 1 mlof 0.9% saline, followed by vortexing for 5 minutes. The lipids werediluted further to the required concentration in the respectiveresuspension vehicles. DM:DP vaccine formulations were prepared at afinal pDNA (phosphate):cationic lipid molar ratio of 4:1 by adding anequal volume of lipid (at twice the final concentration in PBS) to pDNA(at twice the final concentration in PBS). The lipid was added to pDNArapidly as a bolus using a needle and syringe, and after the additionthe vial was inverted several times to produce a uniform suspension.Vaxfectin® vaccine formulations were prepared at a final pDNA(phosphate):cationic lipid molar ratio of 4:1 by adding an equal volumeof lipid (at twice the final concentration in 0.9% saline) to pDNA (attwice the final concentration in 0.9% saline, 20 mM sodium phosphate, pH7.2). The lipid was added in a gentle stream down the side of the pDNAvial, followed by gentle inversion until the suspension was uniform. Allvaccine formulations were prepared on the day of the injection.

Poloxamer 02A (CRL1005+BAK+pDNA) Formulation

Poloxamer 02A (CRL1005+BAK+pDNA) formulations were prepared aspreviously described (Selinsky, C., et al., Hum Vaccine, 1(1), 16-23(2005) and Hartikka, J., et al. J. Gene Medicine, in press (2008)).Briefly, the required concentration of pDNA in PBS (0.9% sodiumchloride+10 mM sodium phosphate, pH 7.2) was stirred on ice and therequired amount of poloxamer CRL1005 was added using a positivedisplacement pipette. The solution was stirred on ice until thepoloxamer dissolved and then the required concentration of BAK dissolvedin PBS was added. The solution was then thermocycled through the cloudpoint (7-12° C.) several times to ensure homogeneity, filter sterilizedthrough a Millipore Steriflip disposable vacuum filtration system(Millipore, Billerica, Mass.) at 4° C. and stored frozen. Prior toinjection, the vaccine was thawed at ambient temperature (25° C.) and,if required, diluted to the required pDNA concentration with PBS abovethe cloud point of CRL1005.

Animal Immunizations

All animal procedures were approved by the Institutional Animal Care andUse Committee (IACUC) and complied with the standards set forth in theGuide for the Care and Use of Laboratory Animals (ILAR, 1996) and theAnimal Welfare Act and Animal Care Regulations (seehttp://www.aphis.usda.gov/ac/publications.html).

For intradermal (ID) immunization with needle & syringe, mice receivedeither two ID injections (20 μl/site) into the dorsal/flank skin, or oneID injection (20 μl/site) into the skin over the gastrocnemius muscle,using a 3/10 cc insulin syringe with a 28G½″ needle.

Epidermal/intradermal immunization of mice was performed usingUltraEnhancer® device (PMT/Permark, Chanhassen, Minn.), which is anoscillating Multi-needle intradermal (OMNI) delivery device. The devicehas been approved in Europe and in the USA for medical and cosmeticmicropigmentation (“tattooing”) applications in humans. Histochemicalanalysis has shown that cells in the upper layers of the dermis and theepidermis get transfected with the antigen-expressing plasmid when pDNAvaccine is delivered to the skin using such a device (Bins, A. D., etal., Nat. Med., 11(8), 899-904 (2005).

OMNI device was used with a sterile 9-needle array (PMT/Permark)consisting of a 3×3 cluster of solid non-injecting steel needles. Thehandpiece of the device was modified by inserting a metal ring betweenthe nose cone and the barrel. Due to this adjustment, penetration depthof the 9-needle array used for mice was limited to approximately 0.5 mm.The approximate diameter of the needle array was 1 mm in each direction.

The immunization procedure was similar to the one previously published(Bins, A. D., et al., Nat. Med., 11(8), 899-904 (2005). Briefly, micewere anesthetized using an appropriate agent such as Isoflurane, and anarea approximately the size of 1 cm² was shaved in the skin over thegastrocnemius muscle of the left leg. The skin was wiped with alcoholand let to dry before vaccination. Twenty microliters of vaccinesolution was applied topically on the skin (single unilateralapplication), immediately followed by treatment with OMNI device for 16seconds with the needle array oscillating at the maximum speed 8,000 RPM(=133 Hz). Approximately 0.5 cm² (0.5 cm wide×1.0 cm long) large area onthe skin was treated by moving the array up and down (1-2 up-and-downmovements/second). Mice were allowed to recover from anesthesia withoutcovering the treated area.

The same procedure was repeated on day 3 and 6 by choosing an adjacentskin area in the left leg. Vaccinations were done in following sequence:lateral (day 0), caudal (day 3), and medial (day 6) face of skin overthe left gastrocnemius muscle. Boost immunizations were administeredinto the contralateral right leg skin on day 21, 24, and 27 in the samesequence: lateral, caudal, and medial face of skin over the rightgastrocnemius muscle.

Immunization of Mice

Groups of 6- to 10-week old female BALB/c mice (Harlan-Sprague-Dawley,Indianapolis, Ind.) received bilateral intramuscular injections into therectusfemoris with pDNA+formulation in 20-50 μl PBS/leg using adisposable 3/10 cc insulin syringe with a 28G½″ needle(Becton-Dickinson, Franklin Lakes, N.J.) as previously described(Hartikka, J., et al., Vaccine, 19(15-16), 1911-23 (2001); Leamy, V. L.,et al., Hum Vaccine, 2(3), 113-8 (2006).

For intradermal (ID) immunization with needle & syringe, mice receivedtwo ID injections (201/site) into the dorsal/flank skin (or the skinover the gastrocnemius muscle) using a 3/10 cc insulin syringe with a28G½″ needle.

Transdermal immunization of mice was performed using UltraEnhancer®device (PMT/Permark, Chanhassen, Minn.), which is an oscillatingmulti-needle intradermal (OMNI) delivery device. The device has beenapproved in Europe and in the USA for medical and cosmeticmicropigmentation (“tattooing”) applications in humans. The device wasused with a sterile 9-needle array (PMT/Permark) consisting of a 3×3cluster of solid non-injecting steel needles. The approximate diameterof the needle array was 1 mm in each direction.

The immunization procedure was similar to the one previously published(Bins, A. D., et al., Nat. Med., 11(8), 899-904 (2005). Briefly, micewere anesthetized using an appropriate agent, and an area approximatelythe size of 1 cm² was shaved in the skin over the gastrocnemius muscleof the left leg. The skin was wiped with alcohol and let to dry beforevaccination. Vaccine solution was applied topically on the skin,immediately followed by treatment with OMNI device for 16 seconds withthe needle array oscillating at the maximum speed 8,000 RPM (=133 Hz).The 9-needle array was adjusted so that the penetration depth was ˜0.5mm. Approximately 0.5 cm² large area in the skin was treated by movingthe array up and down. Mice were allowed to recover from anesthesiawithout covering the treated area. The same procedure was repeated onday 3 and 6 by choosing an adjacent skin area in the left leg.Vaccinations were done in following sequence: lateral, caudal, andmedial face of skin over the left gastrocnemius muscle. Boostimmunizations were administered into the contralateral right leg skin onday 21, 24, and 27 in the same sequence: lateral, caudal, and medialface of skin over the right gastrocnemius muscle.

Mice were bled via the ophthalmic venous plexus at indicated timepoints, and sera were stored at −20° C. until assayed forantigen-specific antibodies by ELISA. Splenocytes were harvested atindicated time points and antigen-specific T-cell responses weremeasured by IFN-γ ELISPOT assay.

Immunization of Rabbits

Female New Zealand White rabbits (5-6 months of age, approximately 2-3kg) were injected in the vastus lateralis muscle with pDNA±formulationin PBS using a 22 gauge 1 inch needle as previously described (Hartikka,J., et al., Vaccine, 19(15-16), 1911-23 (2001); Leamy, V. L., et al.,Hum Vaccine, 2(3), 113-8 (2006).

The needle-free injection device Biojector®2000 (Bioject Incorporated,Portland, Oreg.) is a CO₂ powered jet injection system. The injectionsite was shaved, cleaned with alcohol and the intramuscular injections(vastus lateralis muscle, 500 μL per site) with Biojector®2000 usingBiojector syringe #2 were performed as previously described (Hartikka,J., et al., Vaccine, 19(15-16), 1911-23 (2001). ID vaccinations withBiojector® 2000 were performed in the skin area overlaying vastuslateralis muscle (100 NL per site) using Biojector syringe #2 fittedwith an intradermal spacer. No anesthesia was used for these procedures.

Antibody ELISA Assays

Serum anti-NP IgG titers were measured in an indirect ELISA assay usingninety-six well plates (Corning Incorporated, Corning, N.Y.) coated withinfluenza A/PR/8/34 nucleoprotein (NP) purified from recombinantbaculoviral extracts as previously described (Hartikka, J., et al.,Vaccine, 19(15-16), 1911-23 (2001). Serum anti-M2 IgG titers weremeasured in an indirect ELISA assay using ninety-six well plates coatedwith influenza M2e peptide containing the first 23 amino acids of the M2consensus sequence (MSLLTEVETPRINEWGCRCNDSS (SEQ. ID. NO. 1);Biosynthesis Custom Peptide, Lewisville, Tex.) as previously described(Jimenez, G. S., et al., Hum Vaccine, 3(5), 157-64 (2007). CMVgB-specific IgG antibody responses were measured by ELISA usingninety-six well plates coated with recombinant gB protein (AustralBiologicals, San Ramon, Calif.) as previously described (Selinsky, C.,et al., Hum Vaccine, 1(1), 16-23 (2005). Humoral responses in some serumsamples were analyzed using commercially available Anti-hCMV ELISA kits(BioCheck, San Diego, Calif.). Endpoint titers were determined as thereciprocal of the last dilution at which the absorbance value of thetest serum was at least twice that of the absorbance value of thebackground established with pre-immune serum.

IFN-γ ELISPOT Assays

Mice were sacrificed at indicated time points, splenocytes wereharvested and antigen-specific T-cell responses were measured by IFN-γELISPOT assay as previously described (Selinsky, C., et al., HumVaccine, 1(1), 16-23 (2005). Briefly, ImmunoSpot plates (Millipore,Billerica, Mass.) were coated with rat anti-mouse IFN-γ monoclonalantibody (BD Pharmingen, San Diego, Calif.) and blocked with RPMI-1640medium containing 10% (vol/vol) defined fetal bovine serum (FBS,Hyclone, Logan, Utah). Splenocyte suspensions were seeded inquadruplicate wells of ImmunoSpot plates at 1×10⁶ cells/well inRPMI-1640 medium containing 25 mM HEPES buffer and L-glutamine(Invitrogen, Carlsbad, Calif.) supplemented with 10% (v/v) FBS, 55 μMβ-mercaptoethanol (Invitrogen, Carlsbad, Calif.), 100 U/mL of penicillinG sodium salt and 100 μg/mL of streptomycin sulfate (Invitrogen,Carlsbad, Calif.). For detection of NP-specific IFN-γ secreting CD8+T-cells, splenocytes were stimulated with the NP class I peptide,TYQRTRALV (SEQ. ID. NO. 2), at 4 μg/ml with 1 unit/ml of recombinantmouse IL-2 (Roche, Indianapolis, Ind.). For detection of NP-specificIFN-γ secreting CD4+ T-cells, splenocytes were stimulated with acocktail of three NP class II peptides, FWRGENGKTRSAYERMCNILKGK (SEQ.ID. NO. 3), AVKGVGTMVMELIRMIKRGINDRN (SEQ. ID. NO. 4),RLIQNSLTIERMVLSAFDERRNK (SEQ. ID. NO. 5) at 10 μg/ml each. Afterovernight incubation at +37° C. in 5% CO₂, captured IFN-γ fromstimulated cells was detected by the sequential addition ofbiotin-labeled rat anti-mouse IFN-γ monoclonal antibody (1:1000dilution, BD Pharmingen, San Diego, Calif.) and horseradishperoxidase-labeled avidin D (Vector Labs, Burlingame, Calif.). Spotsproduced by the conversion of 3-amino-9-ethylcarbazole substrate (AEC,Vector Labs, Burlingame, Calif.) were quantified using a C.T.L.ImmunoSpot Analyzer (Cellular Technology Ltd., Cleveland, Ohio). Foreach test group, the average background count derived from unstimulatedcells was subtracted from counts obtained from each well of stimulatedcells. Data were presented as the number of antigen-specific IFN-γproducing T-cells, designated as spot forming units (SFU) per millionsplenocytes (SFU/10⁶ cells).

Influenza Challenge Model

Mice were challenged with 1:10,000 dilution (50 PFU) of mouse-adaptedinfluenza A/HK/68 p#6 as previously described (Jimenez, G. S., et al.,Hum Vaccine, 3(5), 157-64 (2007). For the challenge, mice wereanesthetized with ketamine by i.p. injection and inoculatedintra-nasally with 0.02 ml of virus. Mice were monitored for recoveryafter infection, and then monitored daily for symptoms of disease, lossin body mass and survival through day 21 post-challenge.

Statistical Evaluations

Statistical analysis was performed using either Kruskal-Wallis one-wayANOVA, Student's t-test, or the non-parametric Mann-Whitney rank sumtest (SigmaStat version 2.03, Systat Software, Inc., Point Richmond,Calif.).

1. Intradermal Delivery

Example VIII Cytofectin:co-Lipid Vaccine Formulations Delivered EitherIM or ID with Needle & Syringe Enhance Humoral Immune Responses in Mice

The purpose of the present example (FIG. 1) is to demonstrate theadjuvant effect of Vaxfectin® and DMRIE:DOPE, which was included asanother example of cytofectin:co-lipid adjuvant formulations,administered either IM or ID with needle & syringe in mice.Poloxamer-based formulation, which has been shown to increase theimmunogenicity of pDNA vaccines (Selinsky, C., et al., Hum Vaccine,1(1), 16-23 (2005) and Hartikka, J., et al. J. Gene Medicine, in press(2008)) was also included in the study.

Twelve female BALB/c mice per group were vaccinated with VR4762 pDNAencoding influenza NP antigen. Mice were vaccinated with the followingformulations: 1) pDNA in PBS; 2) Vaxfectin® formulation; 3) DMRIE:DOPE(DM:DP) formulation; and 4) poloxamer 02A formulation. Mice receivedeither bilateral intramuscular (IM) injections into rectus femorismuscle (20 μl/muscle), or two intradermal (ID) injections (20 μl/site)into the dorsal/flank skin with needle & syringe. Mice received 10 μg ofVR4762 per immunization on Day 0 and 21. On day 35, sera were collectedand anti-NP antibody responses were measured using an ELISA assay.

Vaxfectin® and DMRIE:DOPE formulations increased anti-NP titersapproximately 4- and 2-fold, respectively, compared to unformulated pDNAin PBS, both, with 1M and ID injections, and enhanced antibody responsesmore than poloxamer formulation. Furthermore, ID injections of cationiclipid formulations resulted in comparable or higher antibody titers thanwhen the same vaccine formulation was injected IM.

Example IX Vaxfectin®-Formulated pDNA Vaccine Delivered Either IM or IDResults in Stronger Humoral Immune Responses than Unformulated Vaccinein Rabbits

The purpose of the present example (FIG. 2) is to compare IM vs. IDvaccine delivery routes administered either with needle & syringe orwith Biojector®2000 device, and to demonstrate the adjuvant effect ofVaxfectin® formulations. Furthermore, the present example demonstratesthat Vaxfectin® formulations are compatible with a needle-free deliverydevice.

New Zealand White Rabbits (n=6 per group) received either 0.1 mg ofVCL6365 pDNA in PBS or 0.1 mg of Vaxfectin®-formulated VCL6365.Injections were performed either with needle & syringe or with B2000device. On Day 0, rabbits received either a single IM vaccination in theright vastus lateralis muscle (500 μL per muscle), or a single IDvaccination in the skin area overlaying right vastus lateralis (100 μLper site). Identical boost vaccinations were administered on Day 21.Rabbits were bled from the ear vein once a week.

Three- and six-week serum samples were assayed for end-point anti-gBtiters using recombinant gB protein to coat ELISA plates. More detailedtime courses from select experimental groups were analyzed usingcommercially available Anti-hCMV ELISA kits.

Vaxfectin®-formulated vaccine resulted in markedly stronger humoralimmune responses than obtained with unformulated vaccine, both, threeweeks after a single injection (FIG. 2A) and three weeks after boostinjection (FIG. 2B). Furthermore, Vaxfectin® formulation delivered IDwith B2000 device resulted in significantly higher antibody titers thanwhen the same vaccine dose and formulation was delivered IM with B2000,and significantly better responses than when unformulated pDNA vaccinewas injected IM with needle & syringe (FIG. 2C).

Example X Cytofectin:co-Lipid Formulations Result in Stronger HumoralImmune Responses than Obtained with Poloxamer-Based pDNA VaccineFormulations, and Demonstrate a Dose-Sparing Effect in Rabbits

The purpose of the present example (FIG. 3) is to compare humoral immuneresponses obtained in rabbits with cytofectin:co-lipid formulations toresponses obtained with poloxamer-based 02A formulation (poloxamerCRL1005+BAK+pDNA), which previously has been shown to increase theimmunogenicity of pDNA vaccines compared to unformulated pDNA injectedin PBS (Selinsky, C., et al., Hum Vaccine, 1(1), 16-23 (2005) andHartikka, J., et al. J. Gene Medicine, in press (2008)).

New Zealand White Rabbits (n=6 per group) were vaccinated either withpoloxamer 02A (CRL1005+BAK+VCL6365 pDNA) formulations (0.1 mg deliveredeither ID or IM with B2000, or 2.5 mg delivered IM with B2000), or withcytofectin:co-lipid formulations (0.1 mg of VCL6365 formulated withDMRIE:DOPE [D:D] and delivered ID with B2000, or 0.1 mg of VCL6365formulated with Vaxfectin® and injected IM with needle & syringe). OnDay 0, rabbits received either a single 1M vaccination in the rightvastus lateralis muscle (500 μL per muscle), or a single ID vaccinationin the skin area overlaying right vastus lateralis (100 μL per site).Identical boost vaccinations were administered on Day 21. Rabbits werebled from the ear vein once a week.

Six-week serum samples were assayed for end-point anti-gB titers usingrecombinant gB protein to coat ELISA plates. More detailed time coursesfrom select experimental groups were analyzed using commerciallyavailable Anti-hCMV ELISA kits.

When the same dose of pDNA vaccine was delivered ID, DMRIE:DOPEformulation, a representative cytofectin:co-lipid formulation, wassuperior to 02A poloxamer formulation (FIGS. 3A, 3B).

Cytofectin:co-lipid formulations resulted in significantly higherhumoral responses than obtained with the same 0.1 mg dose of 02Apoloxamer formulation injected either ID or 1M, and comparable or higherresponses than obtained when a 25-fold higher dose of 02A formulationwas delivered IM with B2000 device, demonstrating a substantialdose-sparing effect obtained with cytofectin:co-lipid formulationsinjected either with needle & syringe, or delivered with B2000 device(FIGS. 3A, 3B).

Example XI ID Delivery of Vaxfectin® Formulations in Rabbits Result inStronger Antibody Responses than IM Route, and Enable Dose Sparing

The purpose of the present example (FIG. 4) is to compare IM vs. IDimmunization routes in rabbits using Vaxfectin®-formulated pDNA andeither Biojector®2000 device or needle & syringe.

New Zealand White Rabbits (n=6 per group) were immunized with variousdoses of VCL6365 pDNA formulated with Vaxfectin®. The vaccine wasdelivered either IM or ID with Biojector®2000 device. One group ofrabbits was vaccinated with 0.1 mg dose of Vaxfectin®-formulated VCL6365injected IM with needle & syringe. On Day 0, rabbits received either asingle IM vaccination in the right vastus lateralis muscle (500 μL permuscle), or a single ID vaccination in the skin area overlaying rightvastus lateralis (100 μL per site). Identical boost vaccinations wereadministered on Day 21. Rabbits were bled from the ear vein once a week.

Six-week serum samples were assayed for end-point anti-gB titers usingrecombinant gB protein to coat ELISA plates. More detailed time coursesfrom select experimental groups were analyzed using commerciallyavailable Anti-hCMV ELISA kits.

ID delivery of Vaxfectin® formulated pDNA vaccine resulted in strongerhumoral immune responses than when the same formulations were deliveredIM (FIGS. 4A & 4B).

ID delivery of 10 μg of Vaxfectin® formulated pDNA resulted in antibodytiters which were comparable to titers obtained by injecting 100 μg ofVaxfectin® formulated pDNA 1M, demonstrating that ID vaccination routeof Vaxfectin® formulations resulted in a 10-fold dose sparing effect(FIG. 4C).

2. Epidermal/Intradermal (Tattooing) Delivery of Formulated pDNA

Example XII Epidermal/Intradermal Delivery of Vaxfectin®-Formulated pDNAVaccine Results in Better Immune Responses than Obtained with IdInjections Using Needle & Syringe in Mice

The purpose of the present example (FIG. 5) is to compare intradermalinjections with needle & syringe to epidermal/intradermal pDNA vaccinedelivery using OMNI device.

Mice (n=4 per group) were vaccinated with an equal mass of influenzaantigen plasmids VR4759 (M2) and VR4762 (NP). Group A received a totalof 100 μg of unformulated M2/NP pDNA applied topically on the skin (20μl, one site), followed by treatment with OMNI device. Group B receiveda total of 20 μg of M2/NP pDNA formulated with Vaxfectin® appliedtopically on the skin (20 μl, one site), followed by treatment with OMNIdevice. Group C received a total of 20 μg of M2/NP pDNA formulated withVaxfectin® injected intradermally (20 μl, single injection) with needle& syringe. Vaccinations for all groups were done on Day 0, 3, 6, 21, 24,and 27.

On day 35, sera were collected and anti-M2 and anti-NP antibodyresponses were measured using an ELISA assay. On day 48, mice werechallenged with 50 PFU of mouse-adapted influenza A/HK/68 virus, andmonitored thereafter daily for survival and loss in body mass throughday 21 post-challenge.

Humoral immune responses were comparable in group A and B, demonstratinga dose-sparing effect with Vaxfectin® (FIGS. 5A & 5B).

Anti-M2 humoral responses were superior in group B, which receivedVaxfectin®-formulated vaccine epidermally/intradermally with OMNIdevice, compared to group C, which received the same dose of vaccineinjected ID with needle & syringe (FIG. 5A).

Survival in influenza challenge model was better in group B, whichreceived Vaxfectin®-formulated vaccine epidermally/intradermally withOMNI device, compared to group C, which received the same dose ofvaccine injected ID with needle & syringe (FIG. 5C).

Recovery from weight loss was comparable or faster in group B comparedto group A, demonstrating a dose-sparing effect with Vaxfectin® (FIG.5D).

Example XIII Epidermal/Intradermal Delivery of Vaxfectin®-FormulatedpDNA Vaccine Results in Stronger Humoral as Well as Cellular ImmuneResponses, and Better Survival in a Challenge Model, than Obtained withIM or ID Injections Using Needle & Syringe in Mice

The purpose of the present example (FIG. 6) is to compare intramuscularor intradermal injections using needle & syringe toepidermal/intradermal delivery with OMNI device by administering thesame dose of Vaxfectin®-formulated pDNA vaccine.

In group A (n=15), mice were vaccinated with unilateral IM injections(20 μl, right rectus femoris muscle) using needle & syringe. In group B(n=15), mice were vaccinated with unilateral ID injections (20 μl, skinarea over the right gastrocnemius muscle) using needle & syringe. Ingroup C (n=15), mice were vaccinated by applying the vaccine topically(20 μl, one site on the skin over the right gastrocnemius muscle),followed by treatment with OMNI device. All mice were immunized with thesame 20 μg-dose of Vaxfectin®-formulated vaccine (equal mass of NP- andM2-encoding plasmids) administered on Day 0, 3, and 6.

On day 14, one set of mice were challenged with 50 PFU of mouse-adaptedinfluenza A/HK/68 virus, and monitored thereafter daily for survivalthrough day 21 post-challenge (n=10 mice per group). Another set of micewere kept until Day 21, when spleens were harvested to quantitate thenumber of NP-specific IFN-γ-secreting T-cells using an ELISPOT assay,and sera were collected for anti-NP and anti-M2 ELISA assays (n=5 miceper group).

T-cell responses were comparable or stronger when Vaxfectin®-formulatedvaccine was delivered epidermally/intradermally with OMNI device (C)than when injected IM (A) or ID (B) with needle & syringe (FIGS. 6A &6B).

Anti-NP humoral responses were better, and anti-M2 responses weresuperior when Vaxfectin®-formulated vaccine was deliveredepidermally/intradermally with OMNI device (C) than when injected IM (A)or ID (B) with needle & syringe (FIGS. 6C & 6D).

Survival in influenza challenge model was better whenVaxfectin®-formulated vaccine was delivered epidermally/intradermallywith OMNI device (40% protective immunity generated within 2 weeks ingroup C) than when the vaccine was injected IM (A) or ID (B) with needle& syringe (FIG. 6E).

It is to be understood that while the disclosure has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of thedisclosure, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

OTHER REFERENCES

-   Bins, A. D., A. Jorritsma, et al. (2005). “A rapid and potent DNA    vaccination strategy defined by in vivo monitoring of antigen    expression.” Nat Med 11 (8): 899-904.-   Hartikka, J., V. Bozoukova, et al. (2001). “Vaxfectin enhances the    humoral immune response to plasmid DNA-encoded antigens.” Vaccine    19(15-16): 1911-23.-   Hartikka, J., A. Geall, et al. (2008). “Physical Characterization    and in vivo Evaluation of Poloxamer-based DNA Vaccine    Formulations.” J. Gene Medicine: in press.-   Jimenez, G. S., R. Planchon, et al. (2007). “Vaxfectin-formulated    influenza DNA vaccines encoding NP and M2 viral proteins protect    mice against lethal viral challenge.” Hum Vaccine 3(5): 157-64.-   Leamy, V. L., T. Martin, et al. (2006). “Comparison of rabbit and    mouse models for persistence analysis of plasmid-based vaccines.”    Hum Vaccine 2(3): 113-8.-   Selinsky, C., C. Luke, et al. (2005). “A DNA-based vaccine for the    prevention of human cytomegalovirus-associated diseases.” Hum    Vaccine 1(1): 16-23.

1. A compound having formula I:

or an enantiomer, diastereomer, or a pharmaceutically acceptable salt orsolvate thereof, wherein: R¹ and R² are each independently substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl,substituted or unsubstituted heteroarylalkyl, or substituted orunsubstituted carboxyalkyl, wherein R¹ and R² are each optionallyindependently substituted with 1 to 5 R¹⁶ groups; L₁ and L₂ are eachindependently a direct bond, O, NH, N(C₁-C₆ alkyl), or S(O)_(m), whereinm is an integer from 0 to 2; n is an integer from 0 to 6; Q isindependently -Z₁N⁺Z₂- or -Z₁P⁺Z₂-; Z₁ and Z₂ are each independentlyhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl,substituted or unsubstituted arylalkyl, substituted or unsubstitutedheteroarylalkyl, substituted or unsubstituted carboxyalkyl, orsubstituted or unsubstituted —(CH₂)_(m)—R³ wherein m is an integer from1 to 6, and wherein Z₁ and Z₂ are each optionally independentlysubstituted with 1 to 5 R¹⁶ groups; T is substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, or substituted or unsubstitutedcarboxyalkyl; wherein T is optionally independently substituted with 1to 5 R¹⁶ groups; and R³ is independently —OR⁴, —S(O)_(m)R⁵, —NR⁶R⁷,—N⁺R⁸R⁹R¹⁰, —PR¹¹R¹², or —P⁺R¹³R¹⁴R¹⁵; R⁴ and R⁵ are each independentlyhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl,substituted or unsubstituted arylalkyl, substituted or unsubstitutedheteroarylalkyl, or substituted or unsubstituted carboxyalkyl, whereinR⁴ and R⁵ are each optionally independently substituted with 1 to 5 R¹⁶groups; R⁶ and R⁷ are each independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl,substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted carboxyalkyl, or R⁶ and R⁷, together with the N atom towhich they are attached, form substituted or unsubstituted heteroaryl orsubstituted or unsubstituted heterocycloalkyl, wherein R⁶ and R⁷ areeach optionally independently substituted with 1 to 5 R¹⁶ groups; R⁸, R⁹and R¹⁰ are each independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, or substituted or unsubstitutedcarboxyalkyl, or R⁸ is as described, and R⁹ and R¹⁰, together with the Natom to which they are attached, form substituted or unsubstitutedheteroaryl or substituted or unsubstituted heterocycloalkyl, wherein R⁸,R⁹ and R¹⁰ are each optionally independently substituted with 1 to 5 R¹⁶groups; R¹¹, R¹² and R¹³ are each independently substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl,substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted carboxyalkyl, or R¹ is as described, and R¹² and R¹³,together with the P atom to which they are attached, form substituted orunsubstituted heterocycloalkyl, wherein R¹¹, R¹² and R¹³ are eachoptionally independently substituted with 1 to 5 R¹⁶ groups; R¹⁶ ishydrogen, halogen, nitro, cyano, hydroxyl, alkyl, cycloalkyl,perfluoroalkyl, heteroalkyl, heterocycloalkyl, aryl, arylalkyl,heteroaryl, heteroarylalkyl, —(CH₂)_(j)CN,—(CH₂)_(j)OR¹⁷—(CH₂)_(j)C(O)R¹⁷, —(CH₂)_(j)C(O)OR¹⁷, —(CH₂)_(j)NR¹⁸R¹⁹,—(CH₂)_(j)C(O)NR¹⁸R¹⁹, —(CH₂)_(j)OC(O)NR¹⁸R¹⁹, —(CH₂)_(j)NR²⁰C(O)R¹⁷,—(CH₂)_(j)NR²⁰C(O)OR¹⁷, —(CH₂)_(j)N²⁰C(O)NR¹⁸R¹⁹, —(CH₂)_(j)S(O)_(m)R²¹,—(CH₂)_(j)S(O)₂NR¹⁸R¹⁹ or —(CH₂)_(j)NR²⁰S(O)₂R²¹, wherein each j isindependently an integer from 0 to 6, and each m is independently aninteger from 0 to 2; R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹ are each independentlyhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted cycloalkyl, perfluoroalkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted —O-aryl, substitutedor unsubstituted arylalkyl, substituted or unsubstituted heteroaryl,substituted or unsubstituted —O-heteroaryl, or substituted orunsubstituted heteroarylalkyl, or R¹⁷, R²⁰ and R²¹ are as describedabove, and R¹⁸ and R¹⁹, together with the N atom to which they areattached, form substituted or unsubstituted heteroaryl, or substitutedor unsubstituted heterocycloalkyl; with the proviso that the compound offormula I is not GAP-DMORIE; and one or more counter ions.
 2. Anadjuvant composition comprising a mixture of one or more cationic lipidshaving formula I:

or an enantiomer, diastereomer, or a pharmaceutically acceptable salt orsolvate thereof, wherein: R¹ and R² are each independently substitutedor unsubstituted alkyl (includes alkenyl and alkynyl), substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl,substituted or unsubstituted arylalkyl, substituted or unsubstitutedheteroarylalkyl, or substituted or unsubstituted carboxyalkyl, whereinR¹ and R² are each optionally independently substituted with 1 to 5 R⁶groups; L₁ and L₂ are each independently a direct bond, O, NH, N(C₁-C₆alkyl), Se, Te, or S(O)_(m), wherein m is an integer from 0 to 2; n isan integer from 0 to 6; Q is independently -Z₁N⁺Z₂- or -Z₁P⁺Z₂-; Z₁ andZ₂ are each independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl,substituted or unsubstituted arylalkyl, substituted or unsubstitutedheteroarylalkyl, substituted or unsubstituted carboxyalkyl, orsubstituted or unsubstituted —(CH₂)_(m)—R³ wherein m is an integer from1 to 6, and wherein Z₁ and Z₂ are each optionally independentlysubstituted with 1 to 5 R¹⁶ groups; T is substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, or substituted or unsubstitutedcarboxyalkyl; wherein T is optionally independently substituted with 1to 5 R¹⁶ groups; and R³ is independently-OR⁴, —S(O)_(m)R⁵, —NR⁶R⁷,—N⁺R⁸R⁹R¹⁰, —PR¹¹R¹², or —P⁺R¹³R¹⁴R¹⁵; R⁴ and R⁵ are each independentlyhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl,substituted or unsubstituted arylalkyl, substituted or unsubstitutedheteroarylalkyl, or substituted or unsubstituted carboxyalkyl, whereinR⁴ and R⁵ are each optionally independently substituted with 1 to 5 R¹⁶groups; R⁶ and R⁷ are each independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl,substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted carboxyalkyl, or R⁶ and R⁷, together with the N atom towhich they are attached, form substituted or unsubstituted heteroaryl orsubstituted or unsubstituted heterocycloalkyl, wherein R⁶ and R⁷ areeach optionally independently substituted with 1 to 5 R¹⁶ groups; R⁸, R⁹and R¹⁰ are each independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, or substituted or unsubstitutedcarboxyalkyl, or R⁸ is as described, and R⁹ and R¹⁰, together with the Natom to which they are attached, form substituted or unsubstitutedheteroaryl or substituted or unsubstituted heterocycloalkyl, wherein R⁸,R⁹ and R¹⁰ are each optionally independently substituted with 1 to 5 R¹⁶groups; R¹¹, R¹² and R¹³ are each independently substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl,substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted carboxyalkyl, or R¹¹ is as described, and R¹² and R¹³,together with the P atom to which they are attached, form substituted orunsubstituted heterocycloalkyl, wherein R¹¹, R¹² and R¹³ are eachoptionally independently substituted with 1 to 5 R¹⁶ groups; R¹⁶ ishydrogen, halogen, nitro, cyano, hydroxyl, alkyl, cycloalkyl,perfluoroalkyl, heteroalkyl, heterocycloalkyl, aryl, arylalkyl,heteroaryl, heteroarylalkyl, —(CH₂)_(j)CN, —(CH₂)_(j)OR¹⁷,—(CH₂)_(j)C(O)R¹⁷, —(CH₂)_(j)C(O)OR¹⁷, —(CH₂)_(j)NR¹⁷R¹⁸,—(CH₂)_(j)C(O)NR¹⁸R¹⁹, —(CH₂)_(j)OC(O)NR¹⁸R¹⁹, —(CH₂)_(j)NR²⁰C(O)R¹⁷,—(CH₂)_(j)NR²⁰C(O)OR²⁷—(CH₂)_(j)N²⁰C(O)NR¹⁸R¹⁹, —(CH₂)_(j)S(O)_(m)R²¹,—(CH₂)_(j)S(O)₂NR¹⁸R¹⁹, or —(CH₂)_(j)NR²⁰S(O)₂R²¹, wherein each j isindependently an integer from 0 to 6, and each m is independently aninteger from 0 to 2; R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹ are each independentlyhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted cycloalkyl, perfluoroalkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted —O-aryl, substitutedor unsubstituted arylalkyl, substituted or unsubstituted heteroaryl,substituted or unsubstituted —O-heteroaryl, or substituted orunsubstituted heteroarylalkyl, or R¹⁷, R²⁰ and R²¹ are as describedabove, and R¹⁸ and R¹⁹, together with the N atom to which they areattached, form substituted or unsubstituted heteroaryl, or substitutedor unsubstituted heterocycloalkyl; with the proviso that the compound offormula I is not GAP-DMORIE; one or more counter ions, and one or moreco-lipids.
 3. The adjuvant composition of claim 2, wherein: R¹ and R²are each independently substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, or substituted or unsubstituted alkynyl; L₁and L₂ are each independently O or NH; n is 1; and Q is independently-Z₁N⁺Z₂-.
 4. The adjuvant composition of claim 3, wherein R¹ and R² areeach independently substituted or unsubstituted (C₁-C₂₀)alkyl,substituted or unsubstituted (C₂-C₂₀)alkenyl, or substituted orunsubstituted (C₂-C₂₀)alkynyl.
 5. The adjuvant composition of claim 4,wherein R¹ and R² are each independently:


6. The adjuvant composition of claim 3, wherein: Z₁ and Z₂ are eachindependently hydrogen, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl,substituted or unsubstituted arylalkyl, substituted or unsubstitutedheteroarylalkyl, or substituted or unsubstituted carboxyalkyl; and T issubstituted or unsubstituted alkyl.
 7. The adjuvant composition of claim6, wherein: Z₁ and Z₂ are each independently (C₁-C₆)alkyl; and T isindependently (C₁-C₆)alkyl.
 8. The adjuvant composition of claim 3,wherein: Z₁ and Z₂ are each independently substituted or unsubstituted—(CH₂)_(m)—R³; T is substituted or unsubstituted alkyl; and R³ isindependently —NR⁶R⁷ or —N⁺R⁸R⁹R¹⁰.
 9. The adjuvant composition of claim8, wherein: T is independently (C₁-C₆)alkyl; R³ is independently —NR⁶R⁷;and R⁶ and R⁷ are each independently substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, or R⁶ and R⁷, together withthe N atom to which they are attached, form substituted or unsubstitutedheteroaryl or substituted or unsubstituted heterocycloalkyl.
 10. Theadjuvant composition of claim 9, wherein R⁶ and R⁷ are eachindependently substituted or unsubstituted (C₁-C₆)alkyl.
 11. Theadjuvant composition of claim 9, wherein R⁶ and R⁷, together with the Natom to which they are attached, form substituted or unsubstitutedpyrrolyl, substituted or unsubstituted imidazolyl, substituted orunsubstituted pyrrolidinyl, substituted or unsubstituted pyrazolinyl,substituted or unsubstituted piperidinyl, substituted or unsubstitutedpiperazinyl, substituted or unsubstituted morpholinyl, or substituted orunsubstituted thiomorpholinyl.
 12. The adjuvant composition of claim 8,wherein: T is independently (C₁-C₆)alkyl; R³ is independently—N⁺R⁸R⁹R¹⁰; and R⁸, R⁹ and R¹⁰ are each independently substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl, or R⁸ isas described, and R⁹ and R¹⁰, together with the N atom to which they areattached, form substituted or unsubstituted heteroaryl or substituted orunsubstituted heterocycloalkyl.
 13. The adjuvant composition of claim12, wherein R⁸, R⁹ and R¹⁰ are each independently substituted orunsubstituted (C₁-C₆)alkyl.
 14. The adjuvant composition of claim 12,wherein R⁹ and R¹⁰, together with the N atom to which they are attached,form substituted or unsubstituted pyrrolyl, substituted or unsubstitutedimidazolyl, substituted or unsubstituted pyrrolidinyl, substituted orunsubstituted pyrazolinyl, substituted or unsubstituted piperidinyl,substituted or unsubstituted piperazinyl, substituted or unsubstitutedmorpholinyl, or substituted or unsubstituted thiomorpholinyl.
 15. Theadjuvant composition of claim 2, wherein the counter ion is negativelycharged.
 16. The adjuvant composition of claim 15, wherein thenegatively charged counter ion is F⁻, Cl⁻, Br⁻, I⁻, CH₃COO⁻, CF₃COO⁻, orany pharmaceutically acceptable negatively charged counter ion.
 17. Theadjuvant composition of claim 2, wherein the co-lipid is a neutrallipid.
 18. The adjuvant composition of claim 17, wherein the neutrallipid is a phosphatidylethanolamine, and/or a phosphatidylcholine,and/or a mono-, di-, or trialkylglycerol, and/or a mono-, di-, ortriacylglycerol.
 19. The adjuvant composition of claim 18, wherein thephosphatidylethanolamine is1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and/or1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE), and/or1,2-dimyristoyl-glycero-3-phosphoethanolamine (DMPE).
 20. The adjuvantcomposition of claim 19, wherein the phosphatidylethanolamine is1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE).
 21. Theadjuvant composition of claim 2, wherein the compound of formula I andthe co-lipid ratio is from about 9:1 to about 1:9.
 22. The adjuvantcomposition of claim 2, wherein the compound of formula I and theco-lipid are in molar ratio of from about 4:1 to about 1:4.
 23. Theadjuvant composition of claim 2, wherein the compound of formula I andthe co-lipid are in molar ratio of from about 2:1 to about 1:2.
 24. Theadjuvant composition of claim 2, wherein the compound of formula I andthe co-lipid are in molar ratio of about 1:1.
 25. The adjuvantcomposition of claim 2, wherein the compound of formula I and DPyPE arein molar ratio of from about 2:1 to about 1:2.
 26. The adjuvantcomposition of claim 2, wherein the compound of formula I and DPyPE arein molar ratio of about 1:1.
 27. The adjuvant composition of claim 2,wherein the compound of formula I has formulae:


28. A method for immunizing a vertebrate comprising administering into atissue or cavity of the vertebrate an immunogenic composition comprising(a) one or more immunogen-encoding polynucleotide, peptides orpolypeptides, or polysaccharides in an amount sufficient to generate animmune response to the one or more peptides or polypeptides, orpolysaccharides, and (b) the adjuvant composition of claim
 2. 29. Themethod of claim 28, wherein the immunogen-encoding polynucleotide, uponincorporation into the cells of the vertebrate, produces animmunologically effective amount of an immunogen (e.g., an immunogenicprotein).
 30. The method of claim 29, wherein the adjuvant compositionof claim 2 enhances the immune response of the vertebrate to theimmunogen.
 31. A method for immunizing a vertebrate comprisingintradermally administering an immunogenic composition comprising (a)one or more immunogen-encoding polynucleotide, peptides or polypeptides,or polysaccharides in an amount sufficient to generate an immuneresponse to the one or more peptides or polypeptides, orpolysaccharides, and (b) an adjuvant composition.
 32. The method ofclaim 31, wherein the adjuvant composition comprises formula I orformula II and one or more co-lipids.
 33. The method of claim 31,wherein the adjuvant composition comprises GAP-DMORIE and DPyPE.
 34. Acompound having formula II:

or an enantiomer, diastereomer, or a pharmaceutically acceptable salt orsolvate thereof, wherein: R¹ and R² are each independently substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl,substituted or unsubstituted heteroarylalkyl, or substituted orunsubstituted carboxyalkyl, wherein R¹ and R² are each optionallyindependently substituted with 1 to 5 R¹⁶ groups; L₁ and L₂ are eachindependently a direct bond, O, NH, N(C₁-C₆ alkyl), or S(O)_(m), whereinm is an integer from 0 to 2; n is an integer from 1 to 3; Q′ isindependently —N⁺Z₁- or —P⁺Z₁-; Z₁ is independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl, substituted or unsubstitutedarylalkyl, substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted carboxyalkyl, or substituted or unsubstituted—(CH₂)_(m)—R³ wherein m is an integer from 1 to 6, and wherein Z₁ isoptionally independently substituted with 1 to 5 R¹⁶ groups; T issubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl, substituted or unsubstitutedarylalkyl, substituted or unsubstituted heteroarylalkyl, or substitutedor unsubstituted carboxyalkyl; wherein Q is optionally independentlysubstituted with 1 to 5 R¹⁶ groups; and R³ is independently —OR⁴,—S(O)_(m)R⁵, —NR⁶R⁷, —N⁺R⁸R⁹R¹⁰, —PR¹¹R¹², or —P⁺R¹³R¹⁴R¹⁵; R⁴ and R⁵are each independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl,substituted or unsubstituted arylalkyl, substituted or unsubstitutedheteroarylalkyl, or substituted or unsubstituted carboxyalkyl, whereinR⁴ and R⁵ are each optionally independently substituted with 1 to 5 R¹⁶groups; R⁶ and R⁷ are each independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl,substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted carboxyalkyl, or R⁶ and R⁷, together with the N atom towhich they are attached, form substituted or unsubstituted heteroaryl orsubstituted or unsubstituted heterocycloalkyl, wherein R⁶ and R⁷ areeach optionally independently substituted with 1 to 5 R¹⁶ groups; R⁸, R⁹and R¹ are each independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, or substituted or unsubstitutedcarboxyalkyl, or R⁸ is as described, and R⁹ and R¹⁰, together with the Natom to which they are attached, form substituted or unsubstitutedheteroaryl or substituted or unsubstituted heterocycloalkyl, wherein R⁸,R⁹ and R¹⁰ are each optionally independently substituted with 1 to 5 R¹⁶groups; R¹¹, R¹² and R¹³ are each independently substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl,substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted carboxyalkyl, or R¹¹ is as described, and R¹² and R¹³,together with the P atom to which they are attached, form substituted orunsubstituted heterocycloalkyl, wherein R¹¹, R¹² and R¹³ are eachoptionally independently substituted with 1 to 5 R¹⁶ groups; R¹⁶ ishydrogen, halogen, nitro, cyano, hydroxyl, alkyl, cycloalkyl,perfluoroalkyl, heteroalkyl, heterocycloalkyl, aryl, arylalkyl,heteroaryl, heteroarylalkyl, —(CH₂)_(j)CN, —(CH₂)_(j)OR¹⁷,—(CH₂)_(j)C(O)R¹⁷, —(CH₂)_(j)C(O)OR¹⁷, —(CH₂)_(j)NR¹⁸R¹⁹,—(CH₂)_(j)C(O)NR¹⁸R¹⁹, —(CH₂)_(j)OC(O)NR¹⁸R¹⁹, —(CH₂)_(j)NR²⁰C(O)R¹⁷,—(CH₂)_(j)NR²⁰C(O)OR¹⁷, —(CH₂)_(j)N²⁰C(O)NR¹⁸R¹⁹, —(CH₂)_(j)S(O)_(m)R²¹,—(CH₂)_(j)S(O)₂NR¹⁸R¹⁹, or —(CH₂)_(j)NR²⁰S(O)₂R²¹, wherein each j isindependently an integer from 0 to 6, and each m is independently aninteger from 0 to 2; R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹ are each independentlyhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted cycloalkyl, perfluoroalkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted —O-aryl, substitutedor unsubstituted arylalkyl, substituted or unsubstituted heteroaryl,substituted or unsubstituted —O-heteroaryl, or substituted orunsubstituted heteroarylalkyl, or R¹⁷, R²⁰ and R²¹ are as describedabove, and R¹⁸ and R¹⁹, together with the N atom to which they areattached, form substituted or unsubstituted heteroaryl, or substitutedor unsubstituted heterocycloalkyl; and one or more counter ions.